Processes of making and crystalline forms of a mdm2 inhibitor

ABSTRACT

The present invention provides processes for making 2-((3R,5R,6S)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-1-((S)-1-(isopropylsulfonyl)-3-methylbutan-2-yl)-3-methyl-2-oxopiperidin-3-yl)acetic acid as well as intermediates and processes for making the intermediates. Also provided are crystalline forms of the compound and the intermediates.

FIELD OF THE INVENTION

The present invention provides processes for making2-((3R,5R,6S)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-1-((S)-1-(isopropylsulfonyl)-3-methylbutan-2-yl)-3-methyl-2-oxopiperidin-3-yl)aceticacid (“Compound A” herein) as well as intermediates and processes formaking the intermediates. Also provided are crystalline forms of thecompound and the intermediates.

BACKGROUND OF THE INVENTION

p53 is a tumor suppressor and transcription factor that responds tocellular stress by activating the transcription of numerous genesinvolved in cell cycle arrest, apoptosis, senescence, and DNA repair.Unlike normal cells, which have infrequent cause for p53 activation,tumor cells are under constant cellular stress from various insultsincluding hypoxia and pro-apoptotic oncogene activation. Thus, there isa strong selective advantage for inactivation of the p53 pathway intumors, and it has been proposed that eliminating p53 function may be aprerequisite for tumor survival. In support of this notion, three groupsof investigators have used mouse models to demonstrate that absence ofp53 function is a continuous requirement for the maintenance ofestablished tumors. When the investigators restored p53 function totumors with inactivated p53, the tumors regressed.

p53 is inactivated by mutation and/or loss in 50% of solid tumors and10% of liquid tumors. Other key members of the p53 pathway are alsogenetically or epigenetically altered in cancer. MDM2, an oncoprotein,inhibits p53 function, and it is activated by gene amplification atincidence rates that are reported to be as high as 10%. MDM2, in turn,is inhibited by another tumor suppressor, p14ARF. It has been suggestedthat alterations downstream of p53 may be responsible for at leastpartially inactivating the p53 pathway in p53^(WT) tumors (p53wildtype). In support of this concept, some p53^(WT) tumors appear toexhibit reduced apoptotic capacity, although their capacity to undergocell cycle arrest remains intact. One cancer treatment strategy involvesthe use of small molecules that bind MDM2 and neutralize its interactionwith p53. MDM2 inhibits p53 activity by three mechanisms: 1) acting asan E3 ubiquitin ligase to promote p53 degradation; 2) binding to andblocking the p53 transcriptional activation domain; and 3) exporting p53from the nucleus to the cytoplasm. All three of these mechanisms wouldbe blocked by neutralizing the MDM2-p53 interaction. In particular, thistherapeutic strategy could be applied to tumors that are p53^(WT), andstudies with small molecule MDM2 inhibitors have yielded promisingreductions in tumor growth both in vitro and in vivo. Further, inpatients with p53-inactivated tumors, stabilization of wildtype p53 innormal tissues by MDM2 inhibition might allow selective protection ofnormal tissues from mitotic poisons.

The present invention relates to a compound capable of inhibiting theinteraction between p53 and MDM2 and activating p53 downstream effectorgenes. As such, the compound of the present invention would be useful inthe treatment of cancers, bacterial infections, viral infections, ulcersand inflammation. In particular, the compound of the present inventionis useful to treat solid tumors such as: breast, colon, lung andprostate tumors; and liquid tumors such as lymphomas and leukemias. Asused herein, MDM2 means a human MDM2 protein and p53 means a human p53protein. It is noted that human MDM2 can also be referred to as HDM2 orhMDM2.

The compound,2-((3R,5R,6S)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-1-((S)-1-(isopropylsulfonyl)-3-methylbutan-2-yl)-3-methyl-2-oxopiperidin-3-yl)aceticacid, having the chemical structure below

is disclosed in published PCT Application No. WO 2011/153,509 (ExampleNo. 362) This compound, a MDM2 inhibitor, is being investigated in humanclinical trials for the treatment of various cancers. The presentinvention provides processes for making the compound as well asintermediates and processes for making the intermediates. Also providedare crystalline forms of the compound and intermediates.

SUMMARY OF THE INVENTION

In embodiment 1, the present invention provides crystalline

In embodiment 2, the present invention provides crystalline anhydrous

In embodiment 3, the present invention provides crystalline anhydrous

characterized by a powder X-ray diffraction pattern comprising peaks atdiffraction angle 2 theta degrees at approximately 11.6, 12.4, 18.6,19.0, 21.6 and 23.6.

In embodiment 4, the present invention provides crystalline anhydrous

in accordance with claim 3 having the X-ray diffraction patternsubstantially shown in FIG. 1.

In embodiment 5, the present invention provides pharmaceuticalcompositions comprising: crystalline

in accordance with any one of embodiments 1 to 4; and a pharmaceuticallyacceptable excipient.

In embodiment 6, the present invention provides methods of treatingbladder cancer, breast cancer, colon cancer, rectal cancer, kidneycancer, liver cancer, small cell lung cancer, non-small-cell lungcancer, esophagus cancer, gall-bladder cancer, ovarian cancer,pancreatic cancer, stomach cancer, cervix cancer, thyroid cancer,prostate cancer, squamous cell carcinoma, melanoma, acute lymphocyticleukemia, chronic myelogenous leukemia, acute lymphoblastic leukemia,B-cell lymphoma, T-cell-lymphoma, Hodgkin's lymphoma, non-Hodgkin'slymphoma, hairy cell lymphoma, Burkett's lymphoma, acute myelogenousleukemia, chronic myelogenous leukemia, endometrial cancer, head andneck cancer, glioblastoma, osteosarcoma, or rhabdomyosarcoma, themethods comprising administering to a patient in need thereof, atherapeutically acceptable amount of a pharmaceutical compositioncomprising crystalline

in accordance with any one of embodiments 1 to 4.

In embodiment 7, the present invention provides the compound

In embodiment 8, the present invention provides the compound

In embodiment 9, the present invention provides crystalline

In embodiment 10, the present invention provides crystalline

characterized by a powder X-ray diffraction pattern comprising peaks atdiffraction angle 2 theta degrees at approximately 8.7, 18.5, 22.6 and26.6.

In embodiment 11, the present invention provides crystalline

in accordance with claim 10 having the X-ray diffraction patternsubstantially shown in FIG. 3.

In embodiment 12, the present invention provides the compound

In embodiment 13, the present invention provides a process for making

the process comprising:reacting

under dehydrating conditions with

to form

In embodiment 14, the present invention provides the process ofembodiment 13 wherein the dehydrating conditions are azeotropicdistillation with toluene.

In embodiment 15, the present invention provides a process of making

the process comprising:reacting

with

to form

wherein X is CF₃SO₃ ⁻ or

In embodiment 16, the present invention provides a process of making

the process comprising:reacting

with toluene to form

In embodiment 17, the present invention provides a process of making

the process comprising:reacting

with lutidine and

to form

In embodiment 18, the present invention provides a process of making

the process comprising reacting

with

to form

which is oxidized to

which is further converted to

In embodiment 19, the present invention provides a process of embodiment18 wherein the oxidation is accomplished using ozone.

In embodiment 20, the present invention provides a process of embodiment18 wherein the oxidation is accomplished using ozone followed by Pinnickoxidation.

In embodiment 21, the present invention provides a process of embodiment18 wherein the conversion of

is accomplished using methanol and water.

In embodiment 22, the present invention provides a process of embodiment18 wherein the oxidation is accomplished using ozone followed by Pinnickoxidation, and the conversion of

is accomplished using methanol and water.

In embodiment 23, the present invention provides a process of making

the process comprising reacting

with

to form

oxidizing

to form

which is further converted to

In embodiment 24, the present invention provides a process of embodiment23 wherein the

are reacted in the presence of a base.

In embodiment 25, the present invention provides a process of embodiment24 wherein the base is sodium tert-butoxide.

In embodiment 26, the present invention provides a process of embodiment23 wherein the oxidation is accomplished using RuCl₃ and NaIO₄.

In embodiment 27, the present invention provides a process of embodiment23 wherein the conversion of

is accomplished using methanol and water.

In embodiment 28, the present invention provides a process of embodiment23 wherein the

are reacted in the presence of a base;the oxidation is accomplished using RuCl₃ and NaIO₄; andthe conversion of

is accomplished using methanol and water.

In embodiment 29, the present invention provides the compound

In embodiment 30, the present invention provides crystalline

In embodiment 31, the present invention provides crystalline

characterized by a powder X-ray diffraction pattern comprising peaks atdiffraction angle 2 theta degrees at approximately 10.5, 18.2, 20.3, 21,21.9 and 24.2.

In embodiment 32, the present invention provides crystalline

in accordance with embodiment 31 having the X-ray diffraction patternsubstantially shown in FIG. 6.

In embodiment 33, the present invention provides the compound

In embodiment 34, the present invention provides crystalline

In embodiment 35, the present invention provides crystalline

characterized by a powder X-ray diffraction pattern comprising peaks atdiffraction angle 2 theta degrees at approximately 11.5, 14.3, 15.8,17.7, 19.5 and 20.7.

In embodiment 36, the present invention provides crystalline

in accordance with embodiment 35 having the X-ray diffraction patternsubstantially shown in FIG. 12.

In embodiment 37, the present invention provides a process of making

the process comprising reacting

with an oxidizing agent and DABCO to form

and reacting

with an acid to form

In embodiment 38, the present invention provides the process ofembodiment 37 wherein the oxidizing agent is ozone and the acid ishydrochloric acid.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. XRPD Pattern of Compound A Crystalline Anhydrous

FIG. 2. XRPD Pattern of Compound A Amorphous

FIG. 3. XRPD Pattern of Crystalline(3S,5S,6R,8S)-8-allyl-6-(3-chlorophenyl)-5-(4-chlorophenyl)-3-isopropyl-8-methyl-2,3,5,6,7,8-hexahydrooxazolo[3,2-a]pyridin-4-iumnaphthalene-1-sulfonate, hemi-toluene solvate

FIG. 4. XRPD Pattern of Compound A Crystalline Form 1

FIG. 5. XRPD Pattern of Compound A Crystalline Form 2

FIG. 6. XRPD Pattern of Compound A Ethanolate (ethanol solvate)

FIG. 7. XRPD Pattern of Compound A Propanol Solvate

FIG. 8. DSC Curve of Compound A Crystalline Anhydrous

FIG. 9. DSC Curve of Compound A Amorphous

FIG. 10. DSC Curve of Crystalline(3S,5S,6R,85)-8-allyl-6-(3-chlorophenyl)-5-(4-chlorophenyl)-3-isopropyl-8-methyl-2,3,5,6,7,8-hexahydrooxazolo[3,2-a]pyridin-4-iumnaphthalene-1-sulfonate, hemi-toluene solvate

FIG. 11. DSC Curve of Compound A Ethanolate

FIG. 12. XRPD Pattern of Compound A DABCO Salt

FIG. 13. DSC Curve of Compound A DABCO Salt.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides processes for making2-((3R,5R,6S)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-1-((S)-1-(isopropylsulfonyl)-3-methylbutan-2-yl)-3-methyl-2-oxopiperidin-3-yl)aceticacid (“Compound A” herein) as well as intermediates and processes formaking the intermediates. Also provided are crystalline forms of thecompound and the intermediates.

The term “comprising” is meant to be open ended, including the indicatedcomponent but not excluding other elements.

The term “therapeutically effective amount” means an amount of acompound or combination of therapeutically active compounds thatameliorates, attenuates or eliminates one or more symptoms of aparticular disease or condition, or prevents or delays the onset of oneof more symptoms of a particular disease or condition.

The terms “patient” and “subject” may be used interchangeably and meananimals, such as dogs, cats, cows, horses, sheep and humans. Particularpatients are mammals. The term patient includes males and females.

The term “pharmaceutically acceptable” means that the referencedsubstance, such as a compound of the present invention, or a salt of thecompound, or a formulation containing the compound, or a particularexcipient, are suitable for administration to a patient.

The terms “treating”, “treat” or “treatment” and the like includepreventative (e.g., prophylactic) and palliative treatment.

The term “excipient” means any pharmaceutically acceptable additive,carrier, diluent, adjuvant, or other ingredient, other than the activepharmaceutical ingredient (API), which is typically included forformulation and/or administration to a patient.

The compound of the present invention can be administered to a patientin a therapeutically effective amount. The compound can be administeredalone or as part of a pharmaceutically acceptable composition orformulation. In addition, the compound or compositions can beadministered all at once, as for example, by a bolus injection, multipletimes, such as by a series of tablets, or delivered substantiallyuniformly over a period of time, as for example, using transdermaldelivery. It is also noted that the dose of the compound can be variedover time.

The compound of the present invention, or the pharmaceuticallyacceptable salts thereof, may also be administered in combination withone or more additional pharmaceutically active compounds/agents. It isnoted that the additional pharmaceutically active compounds/agents maybe a traditional small organic chemical molecules or can bemacromolecules such as a proteins, antibodies, peptibodies, DNA, RNA orfragments of such macromolecules.

When a patient is to receive or is receiving multiple pharmaceuticallyactive compounds, the compounds can be administered simultaneously, orsequentially. For example, in the case of tablets, the active compoundsmay be found in one tablet or in separate tablets, which can beadministered at once or sequentially in any order. In addition, itshould be recognized that the compositions may be different forms. Forexample, one or more compound may be delivered via a tablet, whileanother is administered via injection or orally as a syrup. Allcombinations, delivery methods and administration sequences arecontemplated.

The term “cancer” means a physiological condition in mammals that ischaracterized by unregulated cell growth. General classes of cancersinclude carcinomas, lymphomas, sarcomas, and blastomas.

The compound of the present invention can be used to treat cancer. Themethods of treating a cancer comprise administering to a patient in needthereof a therapeutically effective amount of the compound, or apharmaceutically acceptable salt thereof.

The compound of the present invention can be used to treat tumors. Themethods of treating a tumor comprise administering to a patient in needthereof a therapeutically effective amount of the compound, or apharmaceutically acceptable salt thereof.

The invention also concerns the use of the compound of the presentinvention in the manufacture of a medicament for the treatment of acondition such as a cancer.

Cancers which may be treated with compounds of the present inventioninclude, without limitation, carcinomas such as cancer of the bladder,breast, colon, rectum, kidney, liver, lung (small cell lung cancer, andnon-small-cell lung cancer), esophagus, gall-bladder, ovary, pancreas,stomach, cervix, thyroid, prostate, and skin (including squamous cellcarcinoma); hematopoietic tumors of lymphoid lineage (includingleukemia, acute lymphocytic leukemia, chronic myelogenous leukemia,acute lymphoblastic leukemia, B-cell lymphoma, T-cell-lymphoma,Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma andBurkett's lymphoma); hematopoietic tumors of myeloid lineage (includingacute and chronic myelogenous leukemias, myelodysplastic syndrome andpromyelocytic leukemia); tumors of mesenchymal origin (includingfibrosarcoma and rhabdomyosarcoma, and other sarcomas, e.g., soft tissueand bone); tumors of the central and peripheral nervous system(including astrocytoma, neuroblastoma, glioma and schwannomas); andother tumors (including melanoma, seminoma, teratocarcinoma,osteosarcoma, xenoderoma pigmentosum, keratoctanthoma, thyroidfollicular cancer and Kaposi's sarcoma). Other cancers that can betreated with the compound of the present invention include endometrialcancer, head and neck cancer, glioblastoma, malignant ascites, andhematopoietic cancers.

Particular cancers that can be treated by the compound of the presentinvention include soft tissue sarcomas, bone cancers such asosteosarcoma, breast tumors, bladder cancer, Li-Fraumeni syndrome, braintumors, rhabdomyosarcoma, adrenocortical carcinoma, colorectal cancer,non-small cell lung cancer, and acute myeleogenous leukemia (AML).

In a particular embodiment of the invention that relates to thetreatment of cancers, the cancer is identified as p53wildtype(p53^(WT)). In another particular embodiment, the cancer is identifiedas p53^(WT) and CDKN2A mutant. In another aspect, the present inventionprovides a diagnostic for determining which patients should beadministered a compound of the present invention. For example, a sampleof a patient's cancer cells may be taken and analyzed to determine thestatus of the cancer cells with respect to p53 and/or CDKN2A. In oneaspect, a patient having a cancer that is p53^(WT) will be selected fortreatment over patients having a cancer that is mutated with respect top53. In another aspect, a patient having a cancer that is both p53^(WT)and has a mutant CDNK2A protein is selected over a patient that does nothave these characteristics. The taking of a cancer cells for analyses iswell known to those skilled in the art. The term “p53^(WT)” means aprotein encoded by genomic DNA sequence no. NC_000017 version 9 (7512445. . . 7531642) (GenBank); a protein encoded by cDNA sequence no.NM_000546 (GenBank); or a protein having the GenBank sequence no.NP_000537.3. The term “CDNK2A mutant” means a CDNK2A protein that is notwildtype. The term “CDKN2A wildtype” means a protein encoded by genomicDNA sequence no. 9:21957751-21984490 (Ensembl ID); a protein encoded bycDNA sequence no. NM_000077 (GenBank) or NM_058195 9GenBank) or; or aprotein having the GenBank sequence no. NP_000068 or NP_478102.

In another aspect, the present invention relates to the use of thecompound of the present invention in combination with one or morepharmaceutical agent that is an inhibitor of a protein in thephosphatidylinositol 3-kinase (PI3K) pathway. Combinations of compoundsof the present invention along with inhibitors of proteins in the PI3Kpathway have shown synergy in cancer cell growth assays, includingenhanced apoptosis and cell killing. Examples of proteins in the PI3Kpathway include PI3K, mTOR and PKB (also known as Akt). The PI3K proteinexists in several isoforms including α, β, δ, or γ. It is contemplatedthat a PI3K inhibitor that can be used in combination with a compound ofthe present invention can be selective for one or more isoform. Byselective it is meant that the compounds inhibit one or more isoformmore than other isoforms. Selectivity is a concept well known to thosein the art and can be measured with well known activity in in vitro orcell-based assays. Preferred selectivity includes greater than 2-fold,preferably 10-fold, or more preferably 100-fold greater selectivity forone or more isoform over the other isoforms. In one aspect, the PI3Kinhibitors that can be used in combination with compounds of the presentinvention is a PI3K a selective inhibitor. In another aspect thecompound is a PI3K 8 selective inhibitor.

Examples of PI3K inhibitors that can be used in combination with one ormore compounds of the present invention include those disclosed in thefollowing: PCT published application no. WO2010/151791; PCT publishedapplication no. WO2010/151737; PCT published application no.WO2010/151735; PCT published application no. WO2010151740; PCT publishedapplication no. WO2008/118455; PCT published application no.WO2008/118454; PCT published application no. WO2008/118468; U.S.published application no. US20100331293; U.S. published application no.US20100331306; U.S. published application no. US20090023761; U.S.published application no. US20090030002; U.S. published application no.US20090137581; U.S. published application no. US2009/0054405; U.S.published application no. U.S. 2009/0163489; U.S. published applicationno. US 2010/0273764; U.S. published application no. U.S. 2011/0092504;or PCT published application no. WO2010/108074.

Compounds that inhibit both PI3K and mTOR (dual inhibitors) are known.In still another aspect, the present invention provides the use of dualPI3K and mTOR inhibitors for use in combination with the compound of thepresent invention.

mTOR is a protein in the PI3K pathway. It is another aspect of thepresent invention to use an mTOR inhibitor in combination with thecompound of the present invention. mTOR inhibitors that can be used incombination with the compound of the present invention include thosedisclosed in the following documents: PCT published application no.WO2010/132598 or PCT published application no. WO2010/096314.

PKB (Akt) is also a protein in the PI3K pathway. It is another aspect ofthe present invention to use an mTOR inhibitor in combination with thecompound of the present invention. PKB inhibitors that can be used incombination with the compound of the present invention include thosedisclosed in the following documents: U.S. Pat. No. 7,354,944; U.S. Pat.No. 7,700,636; U.S. Pat. No. 7,919,514; U.S. Pat. No. 7,514,566; U.S.patent application publication no. US 2009/0270445 A1; U.S. Pat. No.7,919,504; U.S. Pat. No. 7,897,619; or PCT published application no. WO2010/083246 A1.

The combinations of the present invention may also be used inconjunction with radiation therapy, hormone therapy, surgery andimmunotherapy, which therapies are well known to those skilled in theart.

Since one aspect of the present invention contemplates the treatment ofthe disease/conditions with a combination of pharmaceutically activecompounds that may be administered separately, the invention furtherrelates to combining separate pharmaceutical compositions in kit form.The kit comprises two separate pharmaceutical compositions: the compoundof the present invention, and a second pharmaceutical compound. The kitcomprises a container for containing the separate compositions such as adivided bottle or a divided foil packet. Additional examples ofcontainers include syringes, boxes and bags. Typically, the kitcomprises directions for the use of the separate components. The kitform is particularly advantageous when the separate components arepreferably administered in different dosage forms (e.g., oral andparenteral), are administered at different dosage intervals, or whentitration of the individual components of the combination is desired bythe prescribing physician or veterinarian.

An example of such a kit is a so-called blister pack. Blister packs arewell known in the packaging industry and are being widely used for thepackaging of pharmaceutical unit dosage forms (tablets, capsules, andthe like). Blister packs generally consist of a sheet of relativelystiff material covered with a foil of a preferably transparent plasticmaterial. During the packaging process recesses are formed in theplastic foil. The recesses have the size and shape of the tablets orcapsules to be packed. Next, the tablets or capsules are placed in therecesses and the sheet of relatively stiff material is sealed againstthe plastic foil at the face of the foil which is opposite from thedirection in which the recesses were formed. As a result, the tablets orcapsules are sealed in the recesses between the plastic foil and thesheet. Preferably the strength of the sheet is such that the tablets orcapsules can be removed from the blister pack by manually applyingpressure on the recesses whereby an opening is formed in the sheet atthe place of the recess. The tablet or capsule can then be removed viasaid opening.

It may be desirable to provide a memory aid on the kit, e.g., in theform of numbers next to the tablets or capsules whereby the numberscorrespond with the days of the regimen which the tablets or capsules sospecified should be ingested. Another example of such a memory aid is acalendar printed on the card, e.g., as follows “First Week, Monday,Tuesday, . . . etc. . . . Second Week, Monday, Tuesday, . . . ” etc.Other variations of memory aids will be readily apparent. A “daily dose”can be a single tablet or capsule or several pills or capsules to betaken on a given day. Also, a daily dose of a compound of the presentinvention can consist of one tablet or capsule, while a daily dose ofthe second compound can consist of several tablets or capsules and viceversa. The memory aid should reflect this and aid in correctadministration of the active agents. In another specific embodiment ofthe invention, a dispenser designed to dispense the daily doses one at atime in the order of their intended use is provided. Preferably, thedispenser is equipped with a memory-aid, so as to further facilitatecompliance with the regimen. An example of such a memory-aid is amechanical counter which indicates the number of daily doses that hasbeen dispensed. Another example of such a memory-aid is abattery-powered micro-chip memory coupled with a liquid crystal readout,or audible reminder signal which, for example, reads out the date thatthe last daily dose has been taken and/or reminds one when the next doseis to be taken.

The compound of the present invention and other pharmaceutically activecompounds, if desired, can be administered to a patient either orally,rectally, parenterally, (for example, intravenously, intramuscularly, orsubcutaneously) intracisternally, intravaginally, intraperitoneally,intravesically, locally (for example, powders, ointments or drops), oras a buccal or nasal spray. All methods that are used by those skilledin the art to administer a pharmaceutically active agent arecontemplated.

Compositions suitable for parenteral injection may comprisephysiologically acceptable sterile aqueous or nonaqueous solutions,dispersions, suspensions, or emulsions, and sterile powders forreconstitution into sterile injectable solutions or dispersions.Examples of suitable aqueous and nonaqueous carriers, diluents,solvents, or vehicles include water, ethanol, polyols (propylene glycol,polyethylene glycol, glycerol, and the like), suitable mixtures thereof,vegetable oils (such as olive oil) and injectable organic esters such asethyl oleate. Proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preserving,wetting, emulsifying, and dispersing agents. Microorganism contaminationcan be prevented by adding various antibacterial and antifungal agents,for example, parabens, chlorobutanol, phenol, sorbic acid, and the like.It may also be desirable to include isotonic agents, for example,sugars, sodium chloride, and the like. Prolonged absorption ofinjectable pharmaceutical compositions can be brought about by the useof agents delaying absorption, for example, aluminum monostearate andgelatin.

Solid dosage forms for oral administration include capsules, tablets,powders, and granules. In such solid dosage forms, the active compoundis admixed with at least one inert customary excipient (or carrier) suchas sodium citrate or dicalcium phosphate or (a) fillers or extenders, asfor example, starches, lactose, sucrose, mannitol, and silicic acid; (b)binders, as for example, carboxymethylcellulose, alginates, gelatin,polyvinylpyrrolidone, sucrose, and acacia; (c) humectants, as forexample, glycerol; (d) disintegrating agents, as for example, agar-agar,calcium carbonate, potato or tapioca starch, alginic acid, certaincomplex silicates, and sodium carbonate; (a) solution retarders, as forexample, paraffin; (f) absorption accelerators, as for example,quaternary ammonium compounds; (g) wetting agents, as for example, cetylalcohol and glycerol monostearate; (h) adsorbents, as for example,kaolin and bentonite; and (i) lubricants, as for example, talc, calciumstearate, magnesium stearate, solid polyethylene glycols, sodium laurylsulfate, or mixtures thereof. In the case of capsules, and tablets, thedosage forms may also comprise buffering agents. Solid compositions of asimilar type may also be used as fillers in soft and hard filled gelatincapsules using such excipients as lactose or milk sugar, as well as highmolecular weight polyethylene glycols, and the like.

Solid dosage forms such as tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells, such as entericcoatings and others well known in the art. They may also containopacifying agents, and can also be of such composition that they releasethe active compound or compounds in a certain part of the intestinaltract in a delayed manner. Examples of embedding compositions that canbe used are polymeric substances and waxes. The active compound can alsobe in micro-encapsulated form, if appropriate, with one or more of theabove-mentioned excipients.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, and elixirs. Inaddition to the active compounds, the liquid dosage form may containinert diluents commonly used in the art, such as water or othersolvents, solubilizing agents and emulsifiers, as for example, ethylalcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol,dimethylformamide, oils, in particular, cottonseed oil, groundnut oil,corn germ oil, olive oil, castor oil, and sesame seed oil, glycerol,tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid estersof sorbitan, or mixtures of these substances, and the like.

Besides such inert diluents, the composition can also include adjuvants,such as wetting agents, emulsifying and suspending agents, sweetening,flavoring, and perfuming agents. Suspensions, in addition to the activecompound, may contain suspending agents, as for example, ethoxylatedisostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters,microcrystalline cellulose, aluminum metahydroxide, bentonite,agar-agar, and tragacanth, or mixtures of these substances, and thelike.

Compositions for rectal administration are preferable suppositories,which can be prepared by mixing the compounds of the present inventionwith suitable non-irritating excipients or carriers such as cocoabutter, polyethylene glycol or a suppository wax, which are solid atordinary room temperature, but liquid at body temperature, andtherefore, melt in the rectum or vaginal cavity and release the activecomponent.

Dosage forms for topical administration of the compound of the presentinvention include ointments, powders, sprays and inhalants. The activecompound or compounds are admixed under sterile condition with aphysiologically acceptable carrier, and any preservatives, buffers, orpropellants that may be required. Opthalmic formulations, eye ointments,powders, and solutions are also contemplated as being within the scopeof this invention.

The compound of the present invention can be administered to a patientat dosage levels in the range of about 0.1 to about 3,000 mg per day.For a normal adult human having a body weight of about 70 kg, a dosagein the range of about 0.01 to about 100 mg per kilogram body weight istypically sufficient. The specific dosage and dosage range that can beused depends on a number of factors, including the requirements of thepatient, the severity of the condition or disease being treated, and thepharmacological activity of the compound being administered. Thedetermination of dosage ranges and optimal dosages for a particularpatient is within the ordinary skill in the art.

The compound of the present invention can be administered aspharmaceutically acceptable salts, esters, amides or prodrugs. The term“salts” refers to inorganic and organic salts of compounds of thepresent invention. The salts can be prepared in situ during the finalisolation and purification of a compound, or by separately reacting apurified compound in its free base or acid form with a suitable organicor inorganic base or acid and isolating the salt thus formed.Representative salts include the hydrobromide, hydrochloride, sulfate,bisulfate, nitrate, acetate, oxalate, palmitate, stearate, laurate,borate, benzoate, lactate, phosphate, tosylate, citrate, maleate,fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate,lactobionate, and laurylsulphonate salts, and the like. The salts mayinclude cations based on the alkali and alkaline earth metals, such assodium, lithium, potassium, calcium, magnesium, and the like, as well asnon-toxic ammonium, quaternary ammonium, and amine cations including,but not limited to, ammonium, tetramethylammonium, tetraethylammonium,methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine,and the like. See, for example, S. M. Berge, et al., “PharmaceuticalSalts,” J Pharm Sci, 66: 1-19 (1977).

Examples of pharmaceutically acceptable esters of the compound of thepresent invention include C₁-C₈ alkyl esters. Acceptable esters alsoinclude C₅-C₇ cycloalkyl esters, as well as arylalkyl esters such asbenzyl. C₁-C₄ alkyl esters are commonly used. Esters of compounds of thepresent invention may be prepared according to methods that are wellknown in the art.

Examples of pharmaceutically acceptable amides of the compound of thepresent invention include amides derived from ammonia, primary C₁-C₈alkyl amines, and secondary C₁-C₈ dialkyl amines. In the case ofsecondary amines, the amine may also be in the form of a 5 or 6 memberedheterocycloalkyl group containing at least one nitrogen atom. Amidesderived from ammonia, C₁-C₃ primary alkyl amines and C₁-C₂ dialkylsecondary amines are commonly used. Amides of the compound of thepresent invention may be prepared according to methods well known tothose skilled in the art.

The term “prodrug” means compounds that are transformed in vivo to yielda compound of the present invention. The transformation may occur byvarious mechanisms, such as through hydrolysis in blood. A discussion ofthe use of prodrugs is provided by T. Higuchi and W. Stella, “Prodrugsas Novel Delivery Systems,” Vol. 14 of the A.C.S. Symposium Series, andin Bioreversible Carriers in Drug Design, ed. Edward B. Roche, AmericanPharmaceutical Association and Pergamon Press, 1987. To illustrate,because the compound of the invention contains a carboxylic acidfunctional group, a prodrug can comprise an ester formed by thereplacement of the hydrogen atom of the carboxylic acid group with agroup such as (C₁-C₈ alkyl, (C₂—Cl₂)alkanoyloxymethyl,1-(alkanoyloxy)ethyl having from 4 to 9 carbon atoms,1-methyl-1-(alkanoyloxy)ethyl having from 5 to 10 carbon atoms,alkoxycarbonyloxymethyl having from 3 to 6 carbon atoms,1-(alkoxycarbonyloxy)ethyl having from 4 to 7 carbon atoms,1-methyl-1-(alkoxycarbonyloxy)ethyl having from 5 to 8 carbon atoms,N-(alkoxycarbonyl)aminomethyl having from 3 to 9 carbon atoms,1-(N-(alkoxycarbonyl)aminomethyl having from 4 to 10 carbon atoms,3-phthalidyl, 4-crotonolactonyl, gamma-butyrolacton-4-yl,di-N,N—(C₁-C₂)alkylamino(C₂-C₃)alkyl (such as β-dimethylaminoethyl),carbamoyl-(C₁-C₂)alkyl, N,N-di(C₁-C₂)alkylcarbamoyl-(C₁-C₂)alkyl andpiperidino-, pyrrolidino- or morpholino(C₂₋₃)alkyl.

The compound of the present invention may contain asymmetric or chiralcenters, and therefore, exist in different stereoisomeric forms. It iscontemplated that all stereoisomeric forms of the compound as well asmixtures thereof, including racemic mixtures, form part of the presentinvention. In addition, the present invention contemplates all geometricand positional isomers. For example, if the compound contains a doublebond, both the cis and trans forms (designated as Z and E,respectively), as well as mixtures, are contemplated.

Mixture of stereoisomers, such as diastereomeric mixtures, can beseparated into their individual stereochemical components on the basisof their physical chemical differences by known methods such aschromatography and/or fractional crystallization. Enantiomers can alsobe separated by converting the enantiomeric mixture into adiastereomeric mixture by reaction with an appropriate optically activecompound (e.g., an alcohol), separating the diastereomers and converting(e.g., hydrolyzing) the individual diastereomers to the correspondingpure enantiomers. Also, some compounds may be atropisomers (e.g.,substituted biaryls).

The compound of the present invention may exist in unsolvated as well assolvated forms with pharmaceutically acceptable solvents such as water(hydrate), ethanol, and the like. The present invention contemplates andencompasses both the solvated and unsolvated forms as set forth herein.

It is also possible that the compound of the present invention may existin different tautomeric forms. All tautomers of the compound of thepresent invention are contemplated. For example, all of the tautomericforms of the tetrazole moiety are included in this invention. Also, forexample, all keto-enol or imine-enamine forms of the compounds areincluded in this invention.

Those skilled in the art will recognize that the compound names andstructures contained herein may be based on a particular tautomer of acompound. While the name or structure for only a particular tautomer maybe used, it is intended that all tautomers are encompassed by thepresent invention, unless stated otherwise.

It is also intended that the present invention encompass compounds thatare synthesized in vitro using laboratory techniques, such as those wellknown to synthetic chemists; or synthesized using in vivo techniques,such as through metabolism, fermentation, digestion, and the like. It isalso contemplated that the compounds of the present invention may besynthesized using a combination of in vitro and in vivo techniques.

The present invention also includes isotopically-labelled compounds,which are identical to those recited herein, but for the fact that oneor more atoms are replaced by an atom having an atomic mass or massnumber different from the atomic mass or mass number usually found innature. Examples of isotopes that can be incorporated into compounds ofthe invention include isotopes of hydrogen, carbon, nitrogen, oxygen,phosphorous, fluorine and chlorine, such as ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁶O,¹⁷O, ¹⁸O, ³¹P, ³²P, ³⁵S, ¹⁸F, and ³⁶Cl. In one aspect, the presentinvention relates to compounds wherein one or more hydrogen atom isreplaced with deuterium (²H) atoms.

The compound of the present invention that contains the aforementionedisotopes and/or other isotopes of other atoms are within the scope ofthis invention. Certain isotopically-labelled compounds of the presentinvention, for example those into which radioactive isotopes such as ³Hand ¹⁴C are incorporated, are useful in drug and/or substrate tissuedistribution assays. Tritiated, i.e., ³H, and carbon-14, i.e., ¹⁴C,isotopes are particularly preferred for their ease of preparation anddetection. Further, substitution with heavier isotopes such asdeuterium, i.e., ²H, can afford certain therapeutic advantages resultingfrom greater metabolic stability, for example increased in vivohalf-life or reduced dosage requirements and, hence, may be preferred insome circumstances. Isotopically labeled compounds of this invention cangenerally be prepared by substituting a readily available isotopicallylabeled reagent for a non-isotopically labeled reagent.

The compound of the present invention may exist in various solid statesincluding crystalline states and as an amorphous state. The differentcrystalline states, also called polymorphs, and the amorphous states ofthe present compounds are contemplated as part of this invention as setforth herein.

In synthesizing the compound of the present invention, it may bedesirable to use certain leaving groups. The term “leaving groups”(“LG”) generally refer to groups that are displaceable by a nucleophile.Such leaving groups are known in the art. Examples of leaving groupsinclude, but are not limited to, halides (e.g., I, Br, F, Cl),sulfonates (e.g., mesylate, tosylate), sulfides (e.g., SCH₃),N-hydroxsuccinimide, N-hydroxybenzotriazole, and the like. Examples ofnucleophiles include, but are not limited to, amines, thiols, alcohols,Grignard reagents, anionic species (e.g., alkoxides, amides, carbanions)and the like.

All patents, published patent applications and other publicationsrecited herein are hereby incorporated by reference.

The specific experimental examples presented in this applicationillustrate specific embodiments of the present invention. These examplesare meant to be representative and are not intended to limit the scopeof the claims in any manner.

¹H-NMR spectra were typically acquired on a Bruker Avance III 500spectrometer system (Bruker, Billerica, Mass.) operating at a ¹Hfrequency of 500.13 MHz, equipped with a Bruker 5 mm PABBI probe with az-axis gradient; or on a Bruker Avance II or Avance III 400 spectrometeroperating at a ¹H frequency of 400.23 MHz, equipped with a Bruker 5 mmPABBO probe with a z-axis gradient. Samples were typically dissolved in500 μL of either DMSO-d₆ or CD₃OD for NMR analysis. ¹H chemical shiftsare referenced to the residual solvent signals from DMSO-d₆ at δ 2.50and CD₃OD at δ 3.30.

Significant peaks are tabulated and typically include: number ofprotons, multiplicity (s, singlet; d, doublet; dd, doublet of doublets;t, triplet; q, quartet; m, multiplet; br s, broad singlet) and couplingconstant(s) in Hertz. Electron Ionization (EI) mass spectra weretypically recorded on an Agilent Technologies 6140 Quadrupole LC/MS massspectrometer (Agilent Technologies, Englewood, Colo.). Mass spectrometryresults are reported as the ratio of mass over charge, sometimesfollowed by the relative abundance of each ion (in parentheses).Starting materials in the Examples below are typically either availablefrom commercial sources such as Sigma-Aldrich, St. Louis, Mo., or vialiterature procedures.

X-Ray powder diffraction data (XRPD) were obtained using a PANalyticalX'Pert PRO diffractometer (PANalytical, Almelo, The Netherlands) fittedwith a real time multiple strip (RTMS) detector. The radiation used wasCuKα (1.54 Å) and the voltage and current were set at 45 kV and 40 mA,respectively. Data were collected at room temperature from 5 to 45degrees 2-theta with a step size of 0.0334 degrees. Samples wereprepared on a low background sample holder and placed on the samplestage which was rotated with a 2 second revolution time.

Alternatively, XRPD data were obtained using a PANalytical X'Pert PROdiffractometer (PANalytical, Almelo, The Netherlands) fitted with a RTMSdetector. The radiation used was CuKα (1.54 Å) and the voltage andcurrent were set at 45 kV and 40 mA, respectively. Data were collectedat room temperature from 5 to 40, degrees 2-theta with a step size ofeither 0.0334 degrees. Samples were prepared on a low background sampleholder and placed on the sample stage which was rotated with a 2 secondrevolution time.

Alternatively, XRPD data were obtained using a PANalytical X'Pert PROdiffractometer (PANalytical, Almelo, The Netherlands) fitted with a RTMSdetector. The radiation used was CuKα (1.54 Å) and the voltage andcurrent were set at 45 kV and 40 mA, respectively. Data were collectedat room temperature from 5 to 40, degrees 2-theta with a step size ofeither 0.0167 degrees. Samples were prepared on a low background sampleholder and placed on the sample stage which was rotated with a 2 secondrevolution time.

Alternatively, XRPD data were obtained using a PANalytical X'Pert Prodiffractometer (PANalytical, Almelo, The Netherlands) fitted with a RTMSdetector. The radiation used was CuKa (1.54 Å) and the voltage andcurrent were set at 45 kV and 40 mA, respectively. Data were collectedat room temperature from 3 to 40, degrees 2-theta with a step size of0.008 degrees. Samples were prepared on a low background sample holderand placed on the sample stage with a 2 second revolution time.

Alternatively, XRPD data were obtained using a Bruker D8 Discover X-raydiffraction system (Bruker, Billerica, Mass.) fitted with a motorizedxyz sample stage and a GADDS area detector. The radiation used was CuKα(1.54 Å) and the voltage and current were set at 45 kV and 40 mA,respectively. The solid samples on a flat glass plate were mapped andfor each sample an area of 1 mm² was scanned in an oscillating mode for3 minutes from 5 to 48 degrees 2-theta.

Differential Scanning calorimetry (DSC) data was collected usingstandard DSC mode (DSC Q200, TA Instruments, New Castle, Del.). Aheating rate of 10° C./min was employed over a temperature range from40° C. to 300° C. Analysis was run under nitrogen and samples wereloaded in standard, hermetically-sealed aluminum pans. Indium was usedas a calibration standard.

Alternatively, DSC data were collected using temperature-modulated DSCmode (DSC Q200, TA Instruments, New Castle, Del.). After sampleequilibration at 20° C. for five minutes, the heating rate of 3° C./minwas employed with a modulation of +/−0.75° C./min over a temperaturerange from 20° C. to 200° C. Analysis was run under nitrogen and sampleswere loaded in standard, uncrimped aluminum pans. Indium was used as acalibration standard.

The following abbreviations may be used herein.

-   ˜ about-   +ve or pos. ion positive ion-   Δ heat-   Ac acetyl-   ACN acetonitrile-   Ac₂O acetic anhydride-   aq aqueous-   AcOH acetic acid-   Bn benzyl-   Boc tert-butyloxycarbonyl-   BSA bovine serum albumin-   Bu butyl-   Bz benzoyl-   Calcd or Calc'd calculated-   Ca(OH)₂ calcium hydroxide-   CH₃OK potassium methoxide-   CH₃ONa sodium methoxide-   Conc. concentrated-   d day(s)-   DABCO 1,4-diazabicyclo[2.2.2]octane-   DCE dichloroethane-   DCM dichloromethane-   DEA diethylamine-   Dess-Martin periodinane;    1,1,1-triacetoxy-1,1-dihydro-1,2-benziodoxol-3-(1H)-one Dess-Martin    reagent-   DIEA or DIPEA diisopropylethylamine-   DMAP 4-dimethylaminopyridine-   DME 1,2-dimethoxyethane-   DMF N,N-dimethylformamide-   DMSO dimethyl sulfoxide-   DPPA diphenylphosphoryl azide-   dr or DR diastereomeric ratio-   DSC differential scanning calorimetry-   DTT dithiothreitol-   DVB divinylbenzene-   EDC N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide-   ee or e.e. enantiomeric excess-   eq equivalent-   ESI or ES electrospray ionization-   Et ethyl-   Et₂O diethyl ether-   Et₃N triethylamine-   EtOAc ethyl acetate-   EtOH ethyl alcohol-   g gram(s)-   h hour(s)-   HATU O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium    hexafluorophosphate-   HBTU    O-benzotriazole-N,N,N′,N′-tetramethyl-uronium-hexafluorophosphate-   Hex hexanes-   HMPA hexamethylphosphoramide-   HOAt 1-hydroxy-7-azabenzotriazole-   HOBt hydroxybenzotriazole-   HPLC high pressure liquid chromatography-   IPAc or IPAC isopropyl acetate-   IPA or iPrOH isopropyl alcohol-   iPr isopropyl-   Jones reagent solution of chromium(IV)oxide and sulfuric acid in    water-   KHMDS potassium hexamethyldisilazide-   KOAc potassium acetate-   LCMS, LC-MS or LC/MS liquid chromatography mass spectrometry-   LDA lithium diisopropylamide-   LHMDS or LiHMDS lithium hexamethyldisilazide-   L-Selectride® lithium tri-sec-butylborohydride (Sigma-Aldrich, St.    Louis)-   M molar (mol L⁻¹)-   mCPBA m-chloroperoxybenzoic acid-   mDSC modulated differential scanning calorimetry-   Me methyl-   MeCN acetonitrile-   MeI iodomethane-   MEK methyl ethyl ketone-   MeOH methyl alcohol-   mg milligram(s)-   min minute(s)-   mL milliliter(s)-   M mole(s)-   MS mass spectrometry-   MsCl methanesulfonyl chloride-   MTBE or MtBE methyl tert-butyl ether-   m/z mass-to-charge ratio-   NaHMDS sodium hexamethyldisilazide-   NaOtBu sodium tert-butoxide-   NB S N-bromosuccinimide-   nBuLi n-butyl lithium-   NMO N-methylmorpholine-N-oxide-   NMP 1-methyl-2-pyrrolidinone-   NMR nuclear magnetic resonance-   N-Selectride® sodium tri-sec-butylborohydride (Sigma-Aldrich, St.    Louis)-   PBS phosphate buffered saline-   PMB paramethoxybenzyl-   Ph phenyl-   Pr propyl-   ppm parts per million-   PTFE polytetrafluoroethylene-   p-tol para-toluoyl-   rac racemic-   RP-HPLC or RPHPLC reversed phase high pressure liquid chromatography-   RT or rt or r.t. room temperature-   sat. or sat′d or satd saturated-   SFC supercritical fluid chromatography-   TBAF tetrabutylammonium fluoride-   TBDMS tert-butyldimethylsilyl-   TBDMS-Cl tert-butyldimethylsilyl chloride-   TBDPS tert-butyldiphenylsilyl-   TEMPO (2,2,6,6-tetramethylpiperidin-1-yl)oxidanyl-   tert or t tertiary-   TFA trifluoroacetic acid-   TGA thermogravimetric analysis-   THF tetrahydrofuran-   TIPS triisopropylsilyl-   TLC thin layer chromatography-   TMS trimethylsilyl or trimethylsilane-   TPAP tetrapropylammonium perruthenate-   t_(R) retention time-   TRIS 2-amino-2-hydroxymethyl-propane-1,3-diol-   TfOH trifluoroacetic acid-   TfO⁻ trifluoroacetate-   Tf₂O trifluoroacetic acid anhydride-   TsOH or PTSA p-toluenesulfonic acid-   TsO⁻ p-toluenesulfonate-   Ts₂O p-toluenesulfonic acid anhydride-   tBuOH tert-butyl alcohol-   XRD X-ray diffraction-   XRPD or PXRD X-ray powder diffraction-   v/v volume per volume

Procedures to Make Certain Intermediates and Starting Materials Methodfor Making

Step A. 2-(3-Chlorophenyl)-1-(4-chlorophenyl)ethanone

Sodium bis(trimethylsilyl)amide (1 M in tetrahydrofuran, 117 mL) wasslowly added to a −78° C. solution of 2-(3-chlorophenyl) acetic acid (10g, 58.6 mmol) in tetrahydrofuran (58 mL) over 1 hour. After stirring at−78° C. for 40 minutes, a solution of methyl 4-chlorobenzoate (10 g,58.6 mmol) in tetrahydrofuran (35 mL) was added over a period of 10minutes. The reaction was stirred at −78° C. for 3 hours then allowed towarm to 25° C. After two hours at 25° C., the reaction was quenched withsaturated aqueous ammonium chloride solution, and most of thetetrahydrofuran was removed under reduced pressure. The residue wasextracted with ethyl acetate (2×100 mL). The combined organic layerswere washed with saturated sodium chloride solution, dried over sodiumsulfate, filtered and the filtrate was concentrated. The product wasrecrystallized from ether/pentane to provide the title compound as awhite solid.

Alternative Procedure

To a mixture of chlorobenzene (170 L, 1684 mol), 3-chlorophenylaceticacid (50 Kg, 293 mol), and dimethylformamide (0.7 L, 9 mol) at 0° C. wasadded thionyl chloride (39.1 Kg, 329 mol) over the course of 30 min. Themixture was warmed to 15° C. and agitated for 6 h. The mixture wascooled to 0° C. and aluminum chloride (43 Kg, 322 mol) was added overthe course of 1.5 h. The mixture was warmed to 20° C. and agitated for15 h. Water (200 L) and ethanol (200 L) were added to the mixture andthe biphasic mixture was agitated for 2 h. The phases were separated andthe organic phase was washed twice with aqueousethylenediaminetetraacetic acid tetrasodium salt (3 wt %, 200 L), andonce with water (200 L). Heptane (1600 L) was added to the organicphases over the course of 15 minutes. The suspension was agitated for 30minutes, cooled to −5° C., and filtered. The filtered material was driedat 40° C. for 20 h. 2-(3-Chlorophenyl)-1-(4-chlorophenyl)ethanone wasisolated in 83.6% yield (67.4 Kg).

¹H NMR (500 MHz, DMSO-d₆, δ ppm): 8.05 (m, 2H), 7.62 (m, 2H), 7.33 (m,3H), 7.21 (br d, J=7.3 Hz, 1H), 4.45 (s, 2H). MS (ESI)=265.1 [M+H]⁻.

Step B: Methyl4-(3-chlorophenyl)-5-(4-chlorophenyl)-2-methyl-5-oxopentanoate

Methyl methacrylate (12.65 mL, 119 mmol) was added to a solution of2-(3-chlorophenyl)-1-(4-chlorophenyl)ethanone (30 g, 113 mmol) intetrahydrofuran (283 mL). Potassium tert-butoxide (1.27 g, 11.3 mmol)was then added and the reaction was stirred at room temperature for 2days. The solvent was removed under a vacuum and replaced with 300 mL ofethyl acetate. The organic phase was washed with brine (50 mL), water(3×50 mL), and brine (50 mL). The organic phase was dried over magnesiumsulfate, filtered and concentrated under a vacuum to afford methyl4-(3-chlorophenyl)-5-(4-chlorophenyl)-2-methyl-5-oxopentanoate as anapproximately 1:1 mixture of diastereomers.

¹H NMR (400 MHz, CDCl₃, δ ppm): 7.87 (m, 2H), 7.38 (m, 2H), 7.27-7.14(series of m, 4H), 4.61 (m, 1H), 3.69 (s, 1.5H), 3.60 (s, 1.5H), 2.45(m, 1H), 2.34 (m, 1H), 2.10 (ddd, J=13.9, 9.4, 5.5 Hz, 0.5H), 1.96 (ddd,J=13.7, 9.0, 4.3 Hz, 0.5H), 1.22 (d, J=7.0 Hz, 1.5H), 1.16 (d, J=7.0,1.5H). MS (ESI)=387.0 [M+23]⁺.

Step C:(3S,5R,6R)-5-(3-Chlorophenyl)-6-(4-chlorophenyl)-3-methyltetrahydro-2H-pyran-2-oneand(3R,5R,6R)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyltetrahydro-2H-pyran-2-one

Methyl 4-(3-chlorophenyl)-5-(4-chlorophenyl)-2-methyl-5-oxopentanoate(40 g, 104.0 mmol) was dissolved in 200 mL of anhydrous toluene andconcentrated under a vacuum. The residue was placed under high vacuumfor 2 hours before use. The compound was split into 2×20 g batches andprocessed as follows: methyl4-(3-chlorophenyl)-5-(4-chlorophenyl)-2-methyl-5-oxopentanoate (20 g,52.0 mmol) in anhydrous 2-propanol (104 mL) was treated with potassiumtert-butoxide (2.33 g, 20.8 mmol) in a 250 mL glass hydrogenationvessel. RuCl₂(S-xylbinap)(S-DAIPEN) (0.191 g, 0.156 mmol, StremChemicals, Inc., Newburyport, Mass.) in 3.8 mL of toluene was added.After 1.5 hours, the vessel was pressurized to 50 psi (344.7 kPa) andpurged with hydrogen five times and allowed to stir at room temperature.The reaction was recharged with additional hydrogen as needed. After 3days, the reactions were combined and partitioned between 50% saturatedammonium chloride solution and ethyl acetate. The aqueous layer wasextracted with ethyl acetate. The combined organic phases were washedwith brine, dried over magnesium sulfate, filtered, and concentrated.

The crude product (predominantly, (4R,5R)-isopropyl4-(3-chlorophenyl)-5-(4-chlorophenyl)-5-hydroxy-2-methylpentanoate) wasdissolved in tetrahydrofuran (450 mL) and methanol (150 mL). Lithiumhydroxide (1.4 M, 149 mL, 208 mmol) was added, and the solution wasstirred at room temperature for 24 hours. The mixture was concentratedunder a vacuum and the residue was redissolved in ethyl acetate. Aqueous1N hydrochloric acid was added with stirring until the aqueous layer hada pH of about 1. The layers were separated and the organic phase waswashed with brine, dried over magnesium sulfate, filtered andconcentrated. The material was dissolved in 200 mL of anhydrous tolueneand treated with pyridinium p-toluenesulfonate (PPTS, 0.784 g, 3.12mmol). The reaction was heated to reflux under Dean-Stark conditionsuntil the seco-acid was consumed (about 2 hours). The reaction wascooled to room temperature and washed with saturated sodium bicarbonate(50 mL) and brine (50 mL). The solution was dried over sodium sulfate,filtered and concentrated. The crude material was purified by flashchromatography on silica gel (120 g column; eluting with 100%dichloromethane). The title compounds were obtained as a white solidwith an approximate 94:6 enantiomeric ratio and a 7:3 mixture of methyldiastereomers.

¹H NMR (400 MHz, CDCl₃, δ ppm): 7.22-6.98 (series of m, 5H), 6.91 (dt,J=7.4, 1.2 Hz, 0.3H), 6.81 (m, 2H), 6.73 (dt, J=7.6, 1.4 Hz, 0.7H), 5.76(d, J=4.1 Hz, 0.3H), 5.69 (d, J=4.7 Hz, 0.7H), 3.67 (dt, J=6.6, 4.3 Hz,0.3H), 3.55 (td, J=7.8, 4.7 Hz, 0.7H), 2.96 (d of quintets, J=13.5, 6.7Hz, 0.7H), 2.81 (m, 0.3H), 2.56 (dt, J=14.3, 8.0 Hz, 0.7H), 2.32 (dt,J=13.69, 7.0 Hz, 0.3H), 2.06 (ddd, J=13.7, 8.4, 4.1, 0.3H), 1.85 (ddd,J=14.1, 12.5, 7.4, 0.7H), 1.42 (d, J=7.0 Hz, 0.9H), 1.41 (d, J=6.7 Hz,2.1H). MS (ESI)=357.0 [M+23]⁺. [α]_(D) (22° C., c=1.0, CH₂Cl₂)=−31.9°;m.p. 98-99° C.

Step D.(3S,5R,6R)-3-Allyl-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyltetrahydro-2H-pyran-2-one

A solution of(3S,5R,6R)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyltetrahydro-2H-pyran-2-oneand(3R,5S,6S)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyltetrahydro-2H-pyran-2-one(4.5 g, 13.4 mmol) and allyl bromide (3.48 mL, 40.3 mmol) intetrahydrofuran (22 mL) at −35° C. (acetonitrile/dry ice bath) wastreated with a solution of lithium bis(trimethylsilyl)amide intetrahydrofuran (1.0 M, 17.45 mL, 17.45 mmol). The reaction was allowedto warm to −5° C. over 1 hour and then was quenched with 50% saturatedammonium chloride. The reaction was diluted with 100 mL of ethyl acetateand the layers were separated. The organic phase was washed with brine,dried over magnesium sulfate, filtered and concentrated under a vacuumto afford the title compound as a white solid upon standing under avacuum. Chiral SFC (92% CO₂, 8% methanol (20 mM ammonia), 5 mL/min,Phenomenex Lux-2 column (Phenomenex, Torrance, Calif.), 100 bar (10,000kPa), 40° C., 5 minute method) was used to determine that the compoundhad an enantiomeric ratio of 96:4. (Major enantiomer: title compound,retention time=2.45 minutes, 96%; minor enantiomer (structure not shown,retention time=2.12 min, 4%). The title compound was recrystallized byadding to heptane (4.7 g slurried in 40 mL) at reflux and 1.5 mL oftoluene was added dropwise to solubilize. The solution was cooled to 0°C. The white solid was filtered and rinsed with 20 mL of cold heptanesto afford a white powder. Chiral SFC (92% CO₂, 8% methanol, PhenomenexLux-2 column, same method as above) indicated an enantiomeric ratio of99.2:0.8. (major enantiomer, 2.45 min, 99.2%; minor enantiomer: 2.12min, 0.8%)

¹H NMR (400 MHz, CDCl₃, δ ppm): 7.24 (ddd, J=8.0, 2.0, 1.2 Hz, 1H),7.20-7.15 (series of m, 3H), 6.91 (t, J=2.0 Hz, 1H), 6.78 (br d, J=7.6Hz, 1H), 6.60 (m, 2H), 5.84 (ddt, J=17.6, 10.2, 7.4 Hz, 1H), 5.70 (d,J=5.3 Hz, 1H), 5.21-5.13 (series of m, 2H), 3.82 (dt, J=11.7, 4.5 Hz,1H), 2.62 (ABX J_(AB)=13.7 Hz, J_(AX)=7.6 Hz, 1H), 2.53 (ABX,J_(AB)=13.9 Hz, J_(BX)=7.2 Hz, 1H). 1.99 (dd, J=14.1, 11.9 Hz, 1H), 1.92(ddd, J=13.9, 3.9, 1.2 Hz, 1H). ¹³C NMR (CDCl₃, 100 MHz, δ ppm): 175.9,140.2, 134.5, 134.3, 134.0, 132.2, 129.8, 128.6, 128.0, 127.9, 127.8,126.4, 119.9, 83.9, 44.5, 42.4, 40.7, 31.8, 26.1.

MS (ESI)=375.2 [M+H]⁺. IR=1730 cm⁻¹. [α]_(D) (24° C., c=1.0,CH₂Cl₂)=−191°.

m.p. 111-114° C.

Alternative route to make(3S,5R,6R)-3-allyl-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyltetrahydro-2H-pyran-2-one

Step 1: Isopropyl4-(3-chlorophenyl)-5-(4-chlorophenyl)-2-methyl-5-oxopentanoate

A solution of 2-(3-chlorophenyl)-1-(4-chlorophenyl)ethanone (Step A)(67.4 Kg, 255 mol) in THF (325 L) was dried azeotropically to achieve awater content by Karl Fisher of 0.05 wt %. Methyl methacrylate (25.8 Kg,257 mol) was added to the solution and the mixture was heated to 45° C.A solution of potassium tert-butoxide (20 wt % in THF, 14.3 Kg, 25 mol)was added over the course of 30 minutes and the mixture was agitated for6 h. The mixture was cooled to 10° C. and an aqueous solution of citricacid monohydrate (20 wt %, 35 L) was added in less than 5 minutes.Isopropyl acetate (400 L) and an aqueous sodium chloride solution (20 wt%, 300 L) were added. The mixture was agitated for 15 minutes and thephases were separated. The organic phase was distilled under reducedpressure to generate a distillate volume of 560 L while simultaneouslyadding isopropanol (350 L) and producing a solution of methyl4-(3-chlorophenyl)-5-(4-chlorophenyl)-2-methyl-5-oxopentanoate inisopropanol (54 wt %, 140 kg total solution mass). The solution had awater content of 0.01 wt % by Karl Fisher. Additional isopropanol (420L) and sulfuric acid (53 Kg, 535 mol) were added to the solution. Themixture was warmed to reflux and agitated for 12 h, during which time200 L of solvent were distilled and 200 L of fresh isopropanol wereadded to the mixture. The mixture was cooled to 20° C. and water (180 L)was added over the course of 30 minutes. Isopropyl acetate (270 L) wasadded and the mixture was agitated for 30 minutes. The phases wereseparated and the aqueous phase was extracted using isopropyl acetate(100 L). The combined organic phases were washed with water (200 L) fourtimes. The organic phase was distilled under reduced pressure togenerate a distillate volume of 500 L while simultaneously addingisopropanol (50 L) and producing a solution of isopropyl4-(3-chlorophenyl)-5-(4-chlorophenyl)-2-methyl-5-oxopentanoate inisopropanol (60 wt %, 134 kg total solution mass). The solution had awater content of 0.02 wt % by Karl Fisher. The title material wasobtained in 81% overall yield as a roughly 1:1 mixture ifdiastereoisomers.

¹H NMR (400 MHz, CDCl₃, δ ppm): 7.70-7.80 (m, 2H), 7.22-7.28 (m, 2H),7.00-7.18 (series of m, 4H), 4.78-4.96 (m, 1H), 4.42-4.50 (m, 1H),2.02-2.30 (m, 2H), 1.80-1.95 (m, 1H), 0.99-1.19 (m, 15H).

Step 2.(3S,5R,6R)-3-Allyl-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyltetrahydro-2H-pyran-2-one

To a degassed solution of isopropyl4-(3-chlorophenyl)-5-(4-chlorophenyl)-2-methyl-5-oxopentanoate inisopropanol (60 wt %, 252 kg total solution mass, 151 Kg of isopropylester starting material, 385 mol) was added degassed isopropanol (900 L)and potassium tert-butoxide (13 Kg, 116 mol). A separately prepareddegassed solution of (S)-RUCY®-XylBINAP (also known asRuCl[(S)-diapena][(S)-xylbinap] (230 g, 0.2 mol, catalyst, TakasagoInternational Corporation, Rockleigh, N.J.) in isopropanol (25 L). Themixture was purged four times with hydrogen at 5 bars (500 kPa) andagitated at 20° C. for 5.5 h. The hydrogen pressurization wasdiscontinued and the mixture was degassed with nitrogen. Tetrahydrofuran(460 L) was added to the mixture. A solution of lithium hydroxide (24Kg, 576 mol) in water (305 L) was added to the reaction mixture over thecourse of 40 minutes and the resultant mixture was agitated at 20° C.for 24 h. A solution of concentrated hydrochloric acid (79.3 Kg, 11.4 M,740 mol) in water (690 L) was added to the mixture over the course of 2h. Toluene (580 L) was added, the mixture was agitated for 30 minutes,and the phases were separated. The aqueous was extracted using toluene(700 L). The combined organic layers were washed with an aqueoussolution of sodium chloride (25 wt %, 700 Kg). The organic phase wasdistilled at atmospheric pressure and 100° C. to generate a distillatevolume of 2700 L while simultaneously adding toluene (800 L). Less than0.05 wt % isopropanol or water (by Karl Fisher) were left in the mixtureafter this solvent exchange. Carbonyl diimidazole (59 Kg, 365 mol) wasadded to the toluene solution over the course of 2 h and the mixture wasagitated at 20° C. for two additional hours. The mixture was cooled to10° C. and a solution of orthophosphoric acid (72 Kg, 545 mol) in water(400 L) was added over the course of 1 h, while maintaining thetemperature of the mixture below 20° C. The mixture was agitated for 30minutes, the phases were separated and the organic layer was washed withan aqueous solution of sodium chloride (25 wt %, 484 Kg). Toluene (400L) was distilled at atmospheric pressure and 110° C. After cooling ofthe solution to 20° C., tetrahydrofuran (500 L) was added and the watercontent by Karl Fisher was measured to be 0.03 wt %. The productsolution was cooled to −10° C. and a solution allyl bromide (66.8 Kg,552 mol) in tetrahydrofuran (50 L) was added. A lithiumhexamethyldisilazide solution in toluene (255 Kg, 26 wt %, 492 mol) wasadded over the course of 6 h and the mixture was stirred at −10° C. for1 h. The mixture was warmed to 0° C. and an aqueous solution oforthophosphoric acid (40 wt %, 400 mol) was added over the course of 3h. The mixture was warmed to 20° C. Water (200 L) and dichloromethane(400 L) were added. The mixture was agitated for 15 minutes and thephases were separated. The solution was distilled at atmosphericpressure and 100° C. to generate a distillate volume of 1350 L and theresidual toluene in the mixture was measured to be 9.8 wt %. The mixturewas cooled to 70° C. Diisopropyl ether (85 L), water (26 L), andisopropanol (65 L) were added. The mixture was cooled to 35° C.,agitated for 9 h, cooled to 30° C., and filtered. The filtered materialwas washed three times with heptane (80 L). The solids were dried at 55°C. for 48 hours to provide 90.1 Kg of(3S,5R,6R)-3-allyl-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyltetrahydro-2H-pyran-2-onein 63% overall yield. Chiral HPLC indicated an enantiomeric ratio of99.95:0.05.

Step E.(S)-2-((2R,3R)-2-(3-Chlorophenyl)-3-(4-chlorophenyl)-3-hydroxypropyl)-N—((S)-1-hydroxy-3-methylbutan-2-yl)-2-methylpent-4-enamide

(3S,5R,6R)-3-Allyl-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyltetrahydro-2H-pyran-2-one(113 g, 300.0 mmol) was combined with (S)-2-amino-3-methyllbutan-1-ol(93 g, 900.0 mmol) and the suspension was heated at 100° C. for 5 hours.The reaction mixture was cooled to room temperature, diluted with ethylacetate (1000 mL) and washed with 1N hydrochloric acid (2×), water, andbrine. The organic layer was dried over magnesium sulfate andconcentrated under a vacuum to give the title compound as white solidwhich was used in next step without further purification.

Step F.(3S,5S,6R,8S)-8-allyl-6-(3-chlorophenyl)-5-(4-chlorophenyl)-3-isopropyl-8-methyl-2,3,5,6,7,8-hexahydrooxazolo[3,2-a]pyridin-4-iumtrifluoromethanesulfonate

Trifluoromethanesulfonic anhydride (57 mL, 339 mmol) was added dropwiseover 60 minutes via addition funnel to a solution of(S)-2-((2R,3R)-2-(3-chlorophenyl)-3-(4-chlorophenyl)-3-hydroxypropyl)-N—((S)-1-hydroxy-3-methylbutan-2-yl)-2-methylpent-4-enamide(73.7 g, 154 mmol) and 2,6-dimethylpyridine (78 mL, 678 mmol) indichloromethane (700 mL) at −50° C. The reaction mixture was stirred at−50° C. for one additional hour and concentrated under a vacuum toprovide the title compound as a reddish solid which was used in nextstep without further purification.

Step G.(3S,5R,6S)-3-Allyl-5-(3-chlorophenyl)-6-(4-chlorophenyl)-1-((S)-1-(isopropylthio)-3-methylbutan-2-yl)-3-methylpiperidin-2-one

(3S,5S,6R,8S)-8-Allyl-6-(3-chlorophenyl)-5-(4-chlorophenyl)-3-isopropyl-8-methyl-2,3,5,6,7,8-hexahydrooxazolo[3,2-a]pyridin-4-iumtrifluoromethanesulfonate (736 mg, 1.242 mmol) was weighed into an ovendried 50 mL pear-bottom flask and dissolved in 20 mL dry toluene. Thetoluene was removed under a vacuum to remove trace water in the solid.The process was repeated twice, and the resulting residue was driedunder a strong vacuum.

A solution of sodium isopropyl sulfide was prepared by adding potassium2-methylpropan-2-olate (3.0 mL, 3.00 mmol, 1 M solution intetrahydrofuran) to a solution of propane-2-thiol (331 mg, 4.35 mmol) in8 mL dimethylformamide that had been prepared under nitrogen and cooledto 0° C. The sulfide solution was allowed to stir at room temperaturefor five minutes and was cooled to 0° C. The dry(3S,5S,6R,8S)-8-allyl-6-(3-chlorophenyl)-5-(4-chlorophenyl)-3-isopropyl-8-methyl-2,3,5,6,7,8-hexahydrooxazolo[3,2-a]pyridin-4-iumtrifluoromethanesulfonate (736 mg, 1.242 mmol) was dissolved indimethylformamide (8 mL total) and transferred (3 transfers total) viasyringe to the sulfide solution over the course of 5 minutes. After 5minutes, the ice bath was removed and the pale orange solution wasallowed to warm to room temperature.

After stirring overnight, the mixture was partitioned between ethylacetate and saturated ammonium chloride solution. The aqueous phase wassaturated in sodium chloride and back-extracted three times. Thecombined organics were washed twice with saturated sodium bicarbonate,twice with brine, dried over sodium sulfate, filtered, and concentratedunder a vacuum to provide a residue that was purified by silica gelcolumn chromatography (80 g column, gradient elution of 0% to 50% ethylacetate in hexanes).

Method for Making

Step A.(3S,5R,6S)-3-Allyl-5-(3-chlorophenyl)-6-(4-chlorophenyl)-1-((S)-1-hydroxy-3-methylbutan-2-yl)-3-methylpiperidin-2-one

Lithium hydroxide hydrate (64.6 g, 1540 mmol) was added portionwise,over a 5 minute period, to a solution of(3S,5S,6R,85)-8-allyl-6-(3-chlorophenyl)-5-(4-chlorophenyl)-3-isopropyl-8-methyl-2,3,5,6,7,8-hexahydrooxazolo[3,2-a]pyridin-4-iumtrifluoromethanesulfonate (Step F above) dissolved in tetrahydrofuran(500 ml) and water (300 ml). The reaction mixture was stirred at roomtemperature for 1 hour and concentrated under a vacuum. The residue wasdissolved in ethyl acetate (ca. 1.3 L) and the layers were separated.The organic layer was washed with 1N hydrochloric acid (ice cooled, withenough hydrochloric acid to protonate and remove any remaining2,6-dimethylpyridine (300 mL×2)), water and brine. The solvent wasremoved under a vacuum to give a residue which was purified by silicagel column chromatography (1500 g column, gradient elution of 0% to 50%ethyl acetate in hexanes. The product was also crystallized fromcyclohexane.

Step B.(3S,5S,6R,8S)-8-Allyl-6-(3-chlorophenyl)-5-(4-chlorophenyl)-3-isopropyl-8-methyl-2,3,5,6,7,8-hexahydrooxazolo[3,2-a]pyridin-4-ium4-methylbenzenesulfonate

(3S,5R,6S)-3-Allyl-5-(3-chlorophenyl)-6-(4-chlorophenyl)-1-((S)-1-hydroxy-3-methylbutan-2-yl)-3-methylpiperidin-2-one(49.77 g, 98 mmol) was transferred to a 1000 mL flask containing4-methylbenzenesulfonic acid hydrate (19.27 g, 101 mmol) and a stirringbar. The reactants were suspended in toluene (230 mL). The flask wasequipped with a Dean Stark trap and reflux condenser, and the stirredmixture was heated at reflux in a preheated bath. After 1 hour, thesolvent was carefully removed under a vacuum and the resulting residuewas further dried under high vacuum. The title compound was taken to thenext step without purification.

Step C.(3S,5R,6S)-3-Allyl-5-(3-chlorophenyl)-6-(4-chlorophenyl)-1-((S)-1-(isopropylsulfonyl)-3-methylbutan-2-yl)-3-methylpiperidin-2-one

(3S,5S,6R,8S)-8-Allyl-6-(3-chlorophenyl)-5-(4-chlorophenyl)-3-isopropyl-8-methyl-2,3,5,6,7,8-hexahydrooxazolo[3,2-a]pyridin-4-ium4-methylbenzenesulfonate, dry, powdered potassium carbonate (26.9 g, 195mmol) and propane-2-thiol (14 ml, 150 mmol) were added along with 200 mLfreshly sparged dimethylformamide. The mixture was heated under argon at50° C. After about 21 hours, a solution of meta-chloroperbenzoic acid(68.2 g, 77% pure by weight, in 100 mL dimethylformamide) wastransferred to a dropping funnel and rapidly added to the stirredreaction mixture while the flask was immersed in an ice bath. After 5minutes, the resulting yellow solution was allowed to warm to roomtemperature. After 10 minutes, additional meta-chloroperbenzoic acid (12g, 77% wt %) was added as a solid and the mixture was stirred at roomtemperature. Upon completion, the mixture was poured into ethyl acetateand washed with 1 M sodium hydroxide (500 mL) that had been poured intoice. The aqueous phase was back-extracted three times and washed withadditional 1 M NaOH (500 mL, also poured into ice). The aqueous layerwas washed once with ethyl acetate and the organics were combined.Sodium thiosulfate (1 M in water, 250 mL) was added to the organics in alarge Erlenmeyer flask, and the mixture was stirred for twenty minutes.The organic phase was washed again with sodium thiosulfate (1 M inwater, 250 mL) and the mixture was allowed to stand over the weekend.The organics were concentrated to ca. 500 mL, then sequentially washedwith 10% aqueous citric acid, 1 M sodium hydroxide, and brine. Theorganics were dried over sodium sulfate, filtered, and concentrated togive the crude product. The residue was purified by flash columnchromatography (1.5 kg silica gel column, gradient elution of 0% to 50%ethyl acetate in hexanes) to give the title compound as a white solid.

Synthesis of Compound A (Synthesis A)2-((3R,5R,6S)-5-(3-Chlorophenyl)-6-(4-chlorophenyl)-1-((S)-1-(isopropylsulfonyl)-3-methylbutan-2-yl)-3-methyl-2-oxopiperidin-3-yl)aceticacid

Ruthenium(III) chloride trihydrate (22 mg, 0.084 mmol) and sodiumperiodate (1.12 g, 5.24 mmol) were added to a mixture of(3S,5R,6S)-3-allyl-5-(3-chlorophenyl)-6-(4-chlorophenyl)-1-((S)-1-(isopropylthio)-3-methylbutan-2-yl)-3-methylpiperidin-2-one(390 mg, 0.752 mmol) in acetonitrile (4.0 mL), carbon tetrachloride (4.0mL), and water (6.0 mL). The resulting dark brown mixture was vigorouslystirred at ambient temperature overnight. The mixture was filteredthrough a pad of diatomaceous earth, washing with ethyl acetate. Thefiltrate was partitioned between 2 M HCl and ethyl acetate. The aqueousphase was back-extracted twice with ethyl acetate, and the combinedorganics were washed with brine, dried over sodium sulfate, filtered,and concentrated under a vacuum to a residue that was purified by flashchromatography (40 g silica gel column, gradient elution of 0% to 15%isopropanol in hexanes). Fractions containing the desired product werecombined, stripped of solvent, redissolved in minimal ACN/water, frozen,and lyophilized to give a white powder.

Subsequently, a mixture of(3S,5R,6S)-3-allyl-5-(3-chlorophenyl)-6-(4-chlorophenyl)-1-((S)-1-(isopropylthio)-3-methylbutan-2-yl)-3-methylpiperidin-2-one(388 mg, 0.748 mmol), ruthenium(III) chloride trihydrate (19.56 mg,0.075 mmol), and sodium periodate (1.15 g, 5.38 mmol) in acetonitrile (4mL), carbon tetrachloride (4.00 mL), and water (4.00 mL) was vigorouslystirred at ambient temperature. After four hours, the mixture wasfiltered through a pad of diatomaceous earth, and the filtrate waspartitioned between ethyl acetate and 2 M HCl. The aqueous phase wasback-extracted twice with ethyl acetate, and the combined organics werewashed with brine, dried over sodium sulfate, filtered, and concentratedunder a vacuum to a residue. The residue was purified by flashchromatography (40 g silica gel column, gradient elution of 0% to 15%isopropanol in hexanes). Fractions containing the product wereconcentrated and combined with the solid obtained in the priorexperiment. The combined material was dissolved in minimalacetonitrile/water, frozen, and lyophilized overnight to give a whitesolid.

The resulting XRPD pattern was consistent with the amorphous form (FIG.2).

Synthesis of Compound A (Synthesis B)2-((3R,5R,6S)-5-(3-Chlorophenyl)-6-(4-chlorophenyl)-1-((S)-1-(isopropylsulfonyl)-3-methylbutan-2-yl)-3-methyl-2-oxopiperidin-3-yl)aceticacid

Sodium periodate (2.85 g, 13.32 mmol) and ruthenium(III) chloridetrihydrate (0.049 g, 0.189 mmol) were added to a mixture of(3S,5R,6S)-3-allyl-5-(3-chlorophenyl)-6-(4-chlorophenyl)-1-((S)-1-(isopropylsulfonyl)-3-methylbutan-2-yl)-3-methylpiperidin-2-one(1.73 g, 3.14 mmol) in acetonitrile (18 mL), carbon tetrachloride (18mL), and water (27 mL). The mixture was stirred vigorously at roomtemperature for 25 hours. The mixture was diluted with 2M HCl andfiltered through a pad of diatomaceous earth and rinsed with ethylacetate. The organic layer was separated, washed with brine, dried oversodium sulfate, filtered, and concentrated under a vacuum. The materialwas purified twice by flash chromatography (120 g silica gel, gradientelution of 0% to 20% isopropanol in hexanes; 120 g column, gradientelution of 0% to 15% gradient isopropanol in hexanes). It was purifiedonce more by flash chromatography (220 g silica gel; gradient elution 0%to 20% isopropanol in hexanes, 45 minutes) using a method in which thepurest fractions were concentrated and set aside and mixed fractionswere pooled and resubjected to the chromatography.

Subsequently, a mixture of(3S,5R,6S)-3-allyl-5-(3-chlorophenyl)-6-(4-chlorophenyl)-1-((S)-1-(isopropylsulfonyl)-3-methylbutan-2-yl)-3-methylpiperidin-2-one(4.1 g, 7.45 mmol), ruthenium(III) chloride trihydrate (0.120 g, 0.459mmol), and sodium periodate (6.73 g, 31.5 mmol) in acetonitrile (40 mL),carbon tetrachloride (40 mL), and water (60 mL) was vigorously stirredat ambient temperature for 23 hours. The reaction was diluted byaddition of 2 M aqueous HCl and filtered through a diatomaceous earthpad, washing with copious ethyl acetate. Most of the organics wereremoved under a vacuum. The crude product was extracted into ethylacetate, washed with brine, dried over sodium sulfate, filtered, andconcentrated to a residue that was purified twice by flashchromatography (330 g silica gel column, gradient elution of 0% to 20%isopropanol in hexanes; 330 g silica gel column, gradient elution of 0%to 20% isopropanol in hexanes) to give an off-white foam. The materialwas purified by flash chromatography three additional times (220 gsilica gel column; gradient elution 0% to 20% isopropanol in hexanes, 45minutes) using a method in which the purest fractions were concentratedand set aside and mixed fractions were pooled and resubjected to thechromatography.

Mixed fractions from both experiments were combined and purified byflash chromatography twice more (220 g silica gel column; gradientelution 0% to 20% isopropanol in hexanes, 45 minutes), and again thepure fractions were set aside. All of the pure fractions were combined,concentrated under a vacuum, dissolved in minimal acetonitrile/water andlyophilized.

The XRPD pattern was consistent with the amorphous form (FIG. 2).

Synthesis of Compound A (Synthesis C)2-((3R,5R,6S)-5-(3-Chlorophenyl)-6-(4-chlorophenyl)-1-((S)-1-(isopropylsulfonyl)-3-methylbutan-2-yl)-3-methyl-2-oxopiperidin-3-yl)aceticacid

(3S,5R,6S)-3-Allyl-5-(3-chlorophenyl)-6-(4-chlorophenyl)-1-((S)-1-(isopropylsulfonyl)-3-methylbutan-2-yl)-3-methylpiperidin-2-one(5.05 g, 9.17 mmol) was weighed into a 500 mL round bottom flaskcontaining a large stir bar and 2.04 g sodium periodate (2.04 g). Themixture was diluted with carbon tetrachloride (52 mL), acetonitrile, (52mL) and water (78 mL). The flask was immersed in a room temperaturewater bath and the internal temperature was monitored with a digitalthermocouple.

Ruthenium chloride hydrate (approximately 50 mg) was added in a singleportion. The internal temperature rose to 22° C., then ice was added tothe bath to cool the mixture. Additional ruthenium chloride hydrate (25mg) was added 3 minutes later. After stirring for a total of thirtyminutes, Three portions of sodium periodate (2.08 g, 2.07 g and 2.08 g)were slowly added on 15 minute intervals. The temperature was kept below19° C., and ice was quickly added to the bath if the internaltemperature began to rise. The mixture was stirred at ambienttemperature overnight. The mixture was filtered through a pad ofdiatomaceous earth and the filter cake was washed copiously with ethylacetate. The filtrate was concentrated under a vacuum and partitionedbetween 2 M HCl (100 mL) and ethyl acetate (200 mL).

Two rounds of flash column chromatography (330 g silica gel, then 220 gsilica gel, gradient elution of 0% to 20% isopropanol in hexanes)provided the title compound. A portion of this material was lyophilizedfrom acetonitrile and water. The less pure fractions were repurified bytwo additional rounds of flash column chromatography (220 g then 330 gsilica gel columns, gradient elution of 0% to 20% isopropanol inhexanes). The most pure fractions from both runs were combined,concentrated under a vacuum and lyophilized from acetonitrile and waterto give the title compound.

The XRPD pattern was consistent with the amorphous form (FIG. 2).

The three syntheses above resulted in amorphous compound A. Nocrystalline form was obtained. Attempts to crystallize amorphouscompound A made in the above procedure (Synthesis C) are summarized inTable 1A below.

TABLE 1A Mass of Volume Compound solvent A (mg) (mL) Solvent compositionCondition Observation 7.5 1.0 Water/ethanol (90/10 Slurry at Amorphousv/v)) room by XRPD temperature after 2 months 8.0 1.0 Water/dimethylSlurry at Amorphous formamide (90/10 room by XRPD (v/v)) temperatureafter 2 months 8.7 1.0 Heptane/toluene Slurry at Amorphous (98/2 (v/v))room by XRPD temperature after 2 months 8.7 1.0 Heptane/methyl-t- Slurryat Amorphous butylether (98/2 room by XRPD (v/v)) temperature after 2months 9.5 1.0 Cyclohexane/toluene Slurry at Amorphous (98/2 (v/v)) roomby XRPD temperature after 27 days 10.5 1.0 Cyclohexane/methyl- Slurry atAmorphous t-butylether (98/2 room by XRPD (v/v)) temperature after 27days

The amorphous compound made in the procedure above (Synthesis C) wasused in a high-throughput (HT) polymorph screen. The starting materialwas observed to be amorphous by XRPD. In the form screening experiment,of 192 conditions tested, only 1 crystalline sample was observedrepresenting one form shown in FIG. 5 (compound A crystalline Form 2).The form identified by the HTS screen is not consistent with compound Acrystalline anhydrous.

The compound loading amount was about 8 mg/well. Amorphous compound A(Synthesis C) was dispensed into each well on a 96-well glass vial rack.The solid samples in the vials were then transferred to a 96-wellcrystallization source plate.

Per library design, crystallization solvents were dispensed into thesource plate (960 μL/vial) (Table 1 and Table 2). After solventaddition, the source plate was sonicated for 30 minutes, then heated at55° C. with stirring for 30 minutes and kept at 25° C. without stirringfor 30 minutes. Maintaining at 25° C., the solvents in the source platewere aspirated and filtered into a filtration plate. The filtrate wassubsequently aspirated and dispensed into three crystallization plates(evaporation, precipitation, cooling). After completion of 96-wellfiltration, the source plate was kept stirring at 25° C. for 8 hours.The evaporation plate (200 μL/well filtrate) was left open at ambientfor 24 hours. The sealed precipitation plate (150 μL/well filtrateinjected into pre-filled 150 μL anti-solvent; either water or heptane(Table 1)) was cooled linearly from 25° C. to 5° C. in 8 hours and heldat 5° C. for 8 hours. The sealed cooling plate (300 μL/well filtrate)was started at 25° C., cooled to 5° C. in 8 hours, and held at 5° C. foradditional 8 hours. At the end of crystallization, the precipitation andcooling plates were centrifuged at 5° C. for 10 min at 1500 rpm, and thesupernatant in each well of both plates was aspirated and discarded.Prior to dissembling each of 4 plates to collect the crystal samples onits 96-well glass substrates, wick paper was used to dip into each wellto ensure the dryness.

TABLE 1 Solvent Dispense Table For HT Form Screen. All Solvent MixturesAre (V/V). 7 8 9 10 11 12 Anti- Water Water Water Water Heptane Heptanesolvent DCE/Heptane DCE/heptane Toluene/heptane MTBE/heptane THF/heptaneTHF.heptane (5/95) (10/90) (5/95) (5/95) (20/80) (40/60) THF/HeptaneTHF/heptane Toluene/heptane MTBE DMF/heptane DMF/heptane (5/95) (10/90(10/90) (10/90) (20/80) (40/60) IPAc/Heptane IPAc/heptane Acetic acidMEK/heptane Acetone/heptane Acetone/heptane (5/95) (10/90) (5/95)(20/80) (40/60) IPA/Heptane IPA/heptane Heptane MEK/heptaneAcetonitrile/ Acetonitrile/ (5/95) (10/90)e (10/90) heptane heptane(20/80) (40/60) DCE/ DCE/ Toluene/ MTBE/ Ethanol/ Ethanol/ cyclohexanecyclohexane cyclohexane cyclohexane cyclohexane cyclohexane (5/95)(10/90) (5/95) (5/95) (20/80) (40/60) THF/ THF/ Toluene/ MTBE/ IPA/ IPA/cyclohexane cyclohexane cyclohexane cyclohexane cyclohexane cyclohexane(5/95) (10/90) 10/90) (10/90) (20/80) (40/60) IPAc/ IPAc/ Acetic acidMEK/ NMP/ NMP/ cyclohexane cyclohexane cyclohexane cyclohexanecyclohexane (5/95) (10/90) (5/95) (20/80) (40/60) IPA/ IPA/ cyclohexaneMEK/ water 0.01M cyclohexane cyclohexane cyclohexane NaOH in (5/95)(10/90) (10/90) water

Birefringence images were collected for each well of the four 96-wellplates using cross-polarized light optical microscopy. XRPD patternswere collected on a Bruker D8 Discover X-ray diffraction system fittedwith a motorized xyz sample stage and a general area detectordiffraction system (GADDS) area detector. The screen samples on a flatglass plate were mapped and a sample area of 1 mm² was scanned inoscillating mode for 3 minutes from 5° to 48° 20 using CuKa radiation(40 kv, 40 mA) through a graphite monochromator and a collimator of 0.5mm pinhole. In addition to the screen plates the starting material, wasalso analyzed using this instrument and method.

In addition, HT crystallization experiments using bases as additiveswere conducted. Stoichiometric amounts of CH₃OK, CH₃ONa, Tris andammonium hydroxide were added as MeOH solutions, Ca(OH)₂, lysine,diethanolamine, and diethylamine were added as aqueous solutions and thesolvent evaporated under a stream of blown nitrogen prior to solventdispensing.

Per library design, crystallization solvents were dispensed into thesource plate (960 μL/well). After solvent addition, the source plate wassonicated for 30 minutes, then heated at 55° C. with stirring for 30minutes and kept at 25° C. without stirring for 30 minutes. Maintainingat 25° C., the solvents in the source plate were aspirated and filteredinto a filtration plate. The filtrate was subsequently aspirated anddispensed into three crystallization plates (evaporation, precipitation,cooling). After completion of 96-well filtration, the source plate waskept stirring at 25° C. for 8 hours. The evaporation plate (200 μL/wellfiltrate) was left open at ambient for 24 hours. The sealedprecipitation plate (150 μL/well filtrate injected into pre-filled 150μL anti-solvent) was cooled linearly from 25° C. to 5° C. in 8 hours andheld at 5° C. for 8 hours. The sealed cooling plate (300 μL/wellfiltrate) was started at 25° C., cubic cooled to 5° C. in 8 hours, andheld at 5° C. for additional 8 hours. At the end of crystallization, theprecipitation and cooling plates were centrifuged at 5° C. for 10 min at1500 rpm, and the supernatant in each well of both plates was aspiratedand discarded. Prior to dissembling each of 4 plates to collect thecrystal samples on its 96-well glass substrates, wick paper was used todip into each well to ensure the dryness.

None of these experiments resulted in any crystalline salts. Seven (7)samples yielded a crystalline form consistent with the XRPD pattern ofFIG. 4 (compound A crystalline form 1). All crystalline samples observedin this part of the screen were processed by evaporation. Samplesevaporated from IPA with CH₃OK, from MeCN with Tris, from THF/H₂O(90/10) with lysine, from IPA with lysine, from THF/water (90/10) withdiethanolamine, from MeCN with diethanolamine, and from toluene/MeOH(50/50) with diethanol amine gave crystalline samples that wereconsistent with Compound A Crystalline Form 1 by XRPD.

TABLE 2 Solvent Dispense Table For HT Form Screen. All Solvent MixturesAre (V/V). 1 2 3 4 5 6 Counterion\ Heptane Heptane Heptane HeptaneHeptane Heptane anti-solvent A Ammonia THF THF/H₂O IPA MeCN IPAToluene/MeOH (90/10) (50/50) B CH₃OK THF THF/H₂O IPA MeCN IPAToluene/MeOH (90/10) (50/50) C CH₃ONa THF THF/H₂O IPA MeCN IPAToluene/MeOH (90/10) (50/50) D Ca(OH)₂ THF THF/H₂O IPA MeCN IPAToluene/MeOH (0.5 eq) (90/10) (50/50) E Tris THF THF/H₂O IPA MeCN IPAToluene/MeOH (90/10) (50/50) F Lysine THF THF/H₂O EtOH/H₂O MeCN IPAMeCH/H₂O (90/10) (90/10) (90/10) G Dienthanolamine THF THF/H₂O IPA MeCNIPA Toluene/MeOH (90/10) (50/50) H Diethylamine THF THF/H₂O IPA MeCN IPAToluene/MeOH (90/10) (50/50)

Crystallization Studies Experiment 1

2-((3R,5R,6S)-5-(3-Chlorophenyl)-6-(4-chlorophenyl)-1-((S)-1-(isopropylsulfonyl)-3-methylbutan-2-yl)-3-methyl-2-oxopiperidin-3-yl)aceticacid (100 mg) was placed in a 13 mm test tube, and 1 mL of 40% ethanolin water was added at room temperature. The material did not dissolve,even after heating at reflux. An additional 2 mL of 40% ethanol in waterwas added, and still the material did not completely dissolve afterreflux. Ethanol was added dropwise until the material went intosolution. The solution was slowly cooled. The material oiled-out beforereaching room temperature.

Experiment 2

2-((3R,5R,6S)-5-(3-Chlorophenyl)-6-(4-chlorophenyl)-1-((S)-1-(isopropylsulfonyl)-3-methylbutan-2-yl)-3-methyl-2-oxopiperidin-3-yl)aceticacid (100 mg) was placed in a 13 mm test tube and dissolved in 1 mLethanol and heated to reflux. Water was added dropwise until thecloudiness that formed upon addition took a few seconds to disappear (1mL water, total, was added). The solution was cooled slowly. Itoiled-out before reaching room temperature. Additional ethanol (0.2 mL)was added, and the mixture was heated to reflux. The material oiled outupon slow cooling to room temperature. Additional ethanol (0.2 mL) wasadded, and the mixture was heated at reflux. The mixture did not oil outafter cooling to room temperature, but no crystals formed. After 1.5hours at room temperature the solution was placed in the freezer, andthe material oiled-out.

Experiment 3

2-((3R,5R,6S)-5-(3-Chlorophenyl)-6-(4-chlorophenyl)-1-((S)-1-(isopropylsulfonyl)-3-methylbutan-2-yl)-3-methyl-2-oxopiperidin-3-yl)aceticacid (100 mg, white foam) was placed in a 13 mm test tube, and 1 mL of60% ethanol in water was added at room temperature. The foam eithercompletely dissolved or mostly dissolved before precipitating out as awhite solid. The solid was collected by vacuum filtration. Analysisshowed the solid was more pure than the starting material.2-((3R,5R,6S)-5-(3-Chlorophenyl)-6-(4-chlorophenyl)-1-((S)-1-(isopropylsulfonyl)-3-methylbutan-2-yl)-3-methyl-2-oxopiperidin-3-yl)aceticacid (100 mg, white foam) was placed in a 13 mm test tube, and 1 mL of60% ethanol in water was added. The mixture was stirred at roomtemperature during addition and the material briefly dissolved beforeprecipitating as a white solid. The mixture was heated at reflux todissolve the material and slowly cooled to room temperature. Afterstirring overnight at room temperature, no crystals had formed. Thesolution was seeded with solid prepared in the preceding experiment, andsolid formed immediately. The crystals were collected by vacuumfiltration and washed with a cold solution of 60% ethanol in water toprovide a white crystalline solid. Analysis showed further improvementto the purity, and X-ray diffraction indicated the material wascrystalline. The XRPD was consistent with Compound A ethanolate (FIG.6).

Experiment 4

2-((3R,5R,6S)-5-(3-Chlorophenyl)-6-(4-chlorophenyl)-1-((S)-1-(isopropylsulfonyl)-3-methylbutan-2-yl)-3-methyl-2-oxopiperidin-3-yl)aceticacid (100 mg, white foam) was placed in a 13 mm test tube, and 0.75 mLof 60% ethanol in water was added. The mixture was stirred at roomtemperature during addition, and after a few minutes, the foam wasreplaced by a white crystalline solid. The mixture was heated to reflux,slowly cooled to room temperature without stirring. After a few days,large crystals had formed. They were collected by vacuum filtration toprovide the title compound as colorless needles. A single crystal X-raystructure was obtained and was consistent with compound A ethanolate(FIG. 6).

Synthesis of Compound A Ethanolate2-((3R,5R,6S)-5-(3-Chlorophenyl)-6-(4-chlorophenyl)-1-((S)-1-(isopropylsulfonyl)-3-methylbutan-2-yl)-3-methyl-2-oxopiperidin-3-yl)aceticacid

(3S,5R,6S)-3-Allyl-5-(3-chlorophenyl)-6-(4-chlorophenyl)-1-((S)-1-(isopropylsulfonyl)-3-methylbutan-2-yl)-3-methylpiperidin-2-one(86.8 g, 158 mmol) was dissolved in acetonitrile (300 mL) and ethylacetate (300 mL) and transferred to a 2 L 3-neck Morton flask. Water(450 mL) was added. The flask was equipped with a thermocouple andmagnetic stir bar and then submerged in a water bath. Ruthenium(III)chloride hydrate (0.782 g, 3.47 mmol) was added followed by sodiumperiodate (33.75 g). The temperature rose from 17° C. to 22° C. After 35minutes, a second aliquot of sodium periodate (33.75 g) was added andthe temperature increased from 21° C. to 25° C. After 38 minutes, athird aliquot of sodium periodate (33.75 g) was added, and thetemperature increased from 22° C. to 28° C. over 12 minutes. Ice wasadded to the water bath and once the mixture had cooled (approximately 8minutes) a third aliquot of sodium periodate (35 g) was added. Thetemperature increased from 21° C. to 25° C. After stirring at roomtemperature overnight, sodium periodate (20 g) was added, and 4 hourslater, another aliquot of sodium periodate (20 g) was added. After onehour, the mixture was stirred at room temperature with an overheadstirrer. Then the reaction mixture was filtered through a Büchner funneland the filter cake was rinsed with ethyl acetate. The cake was driedovernight in the vacuum filtration apparatus.

The material was added to a large separatory funnel with water (1 L) andethyl acetate (500 mL). Brine was added (50 mL). After 5 hours, thephases were separated and the organic phase was washed with 10% sodiumbisulfite solution. After standing overnight, the phases were separatedand the organic phase was washed with brine (1 L). After 30 minutes theorganic phase was separated, dried over sodium sulfate, filtered andconcentrated under a vacuum. The crude material was purified by flashcolumn chromatography (1.5 kg silica gel column, gradient elution of 0%to 50% isopropanol in hexanes) to provide the title compound as a whitefoam.

The resulting2-((3R,5R,6S)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-1-((S)-1-(isopropylsulfonyl)-3-methylbutan-2-yl)-3-methyl-2-oxopiperidin-3-yl)aceticacid was dissolved in ethanol and transferred to a 500 mL pear shapedflask. The solvent was removed under a vacuum to provide a white solid.A solution of 60% ethanol in water (360 mL) was added and the mixturewas heated to 90° C. to dissolve all of the material. The solution wasslowly cooled and seeded at 50° C., 45° C., and 40° C. withapproximately 5 mg of crystalline product but the material dissolved.The solution was seeded at 37° C. with approximately 5 mg crystallineproduct and the material did not dissolve. The material was slowlycooled to room temperature and placed in the freezer overnight. Crystalswere collected by vacuum filtration through a Büchner funnel and washedwith cold 60% ethanol in water (approximately 100 mL). The material wasdried by pulling air through the filter bed for 4 hours to provide awhite solid (80.6 g). The material was placed under a vacuum at roomtemperature for two days. Next, the material was placed on a rotaryevaporator at 50° C. at 15 Torr (2 kPa) for 4 hours. It was then placedunder a vacuum at 50° C. overnight. NMR analysis indicated 6 wt %ethanol was present in the sample.

A small portion of the sample (100 mg) was slurried in water (0.5 mL)overnight. The solid was collected by vacuum filtration and washed withwater to provide a white solid. NMR analysis indicated that 2.9 wt %ethanol was present. The material was re-slurried in water (0.5 mL)overnight and collected by vacuum filtration to provide a white solid.NMR analysis indicated that 0.5 wt % ethanol was present. X-raydiffraction indicated that the material had become amorphous.

The remainder of the material was heated at 55° C. under a vacuumovernight. After cooling to room temperature, it was slurried in water(250 mL) and stirred mechanically. Aliquots were periodically removedand the solid was measured for ethanol content. After 40 hours,additional water (100 mL) was added and the material was stirred at roomtemperature for an additional 4.5 days. The material was collected byvacuum filtration to provide a white granular solid which wasresuspended in water (350 mL) and mechanically stirred at roomtemperature for about 8 hours. The material was collected by vacuumfiltration through a Büchner funnel to provide a white solid. The solidwas dried by pulling air through the filter bed for 6 hours and then itwas allowed sit open to the atmosphere in the hood overnight to providea white solid containing 3.5 wt % ethanol.

Manual Polymorph Screening

Samples were prepared according to the following general procedure.Approximately 20 mg of compound A ethanolate were weighted and added toa 1 dram vial. Solvent, 1 mL, was added to the vial. The samples wereallowed to slurry. Solvents tested were water/ethanol (80/20, v/v),water/ethanol (70/30, v/v), water/ethanol (60/40, v/v), water/l-propanol(90/10, v/v), water/l-propanol (80/20, v/v), water/1-propanol (70/30,v/v), water/acetonitrile (95/5, v/v), water/acetonitrile (90/10, v/v),water/acetone (95/5, v/v), water/acetone (90/10, v/v), heptane,heptane/isopropyl acetate (99/1, v/v), cyclohexane,cyclohexane/isopropyl acetate (99/1, v/v). Observations were noted atthe start of the experiment and on days 3, 7, 10, 13 and 19. Sampleswere analyzed by XRPD on days 7 and 10, 13, or 19. Results are given inTable 3. The XRPD was consistent with Compound A ethanolate (FIG. 6),Compound A Propanol Solvate (FIG. 7), Compound A Crystalline Anhydrous(FIG. 1) or Compound A Amorphous (FIG. 2).

TABLE 3 Sample No. Solvent Crystalline Form 1 water/ethanol (80/20, v/v)Yes Ethanolate 2 water/ethanol (70/30, v/v) Yes Ethanolate 3water/ethanol (60/40, v/v) Yes Ethanolate 4 water/1-propanol (90/10, YesPropanol v/v) Solvate 5 water/1-propanol (80/20, Yes Propanol v/v)Solvate 6 water/1-propanol (70/30, Yes Propanol v/v) Solvate 7water/acetonitrile (95/5, Yes Crystalline v/v) Anhydrous 8water/acetonitrile (90/10, Yes Crystalline v/v) Anhydrous 9water/acetone (95/5, v/v) Yes Crystalline Anhydrous 10 water/acetone(90/10, v/v) No Amorphous 11 heptane Yes Crystalline Anhydrous 12heptane/isopropyl acetate Yes Crystalline (99/1, v/v) Anhydrous 13cyclohexane Yes Crystalline Anhydrous 14 cyclohexane/isopropyl YesCrystalline acetate (99/1, v/v) Anhydrous

Synthesis of Compound A Ethanolate2-((3R,5R,6S)-5-(3-Chlorophenyl)-6-(4-chlorophenyl)-1-((S)-1-(isopropylsulfonyl)-3-methylbutan-2-yl)-3-methyl-2-oxopiperidin-3-yl)aceticacid

A number of batches were processed in series and combined for the finalpurification.

Batch 1

(3S,5R,6S)-3-Allyl-5-(3-chlorophenyl)-6-(4-chlorophenyl)-1-((S)-1-(isopropylsulfonyl)-3-methylbutan-2-yl)-3-methylpiperidin-2-one(80.6 g, 146 mmol) was dissolved in acetonitrile (280 mL) and ethylacetate (280 mL) and transferred to a 2 L 3-neck Morton flask. Water(418 mL) was added. The flask was equipped with a thermocouple andsubmerged in a water bath. Ruthenium(III) chloride hydrate (0.726 g,3.22 mmol) was added followed by sodium periodate (31.25 g). Thetemperature rose from 17° C. to 24° C., and ice was added to a waterbath to control the temperature. After 15 minutes, a second aliquot ofsodium periodate (31.25 g) was added and the temperature increased from18° C. to 20° C. After 15 minutes a third aliquot of sodium periodate(31.25 g) was added and the temperature increased from 18° C. to 25.6°C. Additional ice was added to the water bath. After 10 minutes, afourth aliquot of sodium periodate (31.25 g) was added. After stirringfor two hours sodium periodate was added (15 g) and after 90 minutessodium periodate (6 g) was added again. After one hour, the liquid wasdecanted into a large separatory funnel. The solid material was rinsedwith ethyl acetate (1.5 L), added to the separatory funnel, and washedwith 10% sodium bisulfite (1 L). The organic layer was washed with brineand the phases were allowed to separate overnight. The solid materialwas re-slurried with ethyl acetate (300 mL) and filtered. The filtratewas washed with 10% sodium bisulfite and brine. The combined organiclayers were dried over sodium sulfate, filtered, and concentrated. Thecrude material was purified by flash column chromatography (1.5 kgsilica gel column, gradient elution of 0% to 50% isopropanol in hexanes)to provide the title compound.

Batch 2

(3S,5R,6S)-3-Allyl-5-(3-chlorophenyl)-6-(4-chlorophenyl)-1-((S)-1-(isopropylsulfonyl)-3-methylbutan-2-yl)-3-methylpiperidin-2-one(90.4 g, 162 mmol) was dissolved in acetonitrile (308 mL) and ethylacetate (308 mL) and transferred to a 2 L 3-neck Morton flask. Water(463 mL) was added. The flask was equipped with a thermocouple and amechanical stirrer. Ruthenium(III) chloride hydrate (0.803 g, 3.56 mmol)was added and the reaction vessel was submerged in a cool water bath.Sodium periodate was added in portions (first portion: 34.0 g), and thetemperature was monitored to keep the reaction mixture below 25° C. Icewas periodically added to the water bath to assist in temperaturecontrol.

After stirring for 12 minutes, a second portion was added (39.7 g),followed 28 minutes later by a third portion (36.6 g), and after 13minutes, a forth portion (35.6 g). The mixture was stirred overnight atroom temperature, and a fifth portion was added (15 g), and after 25minutes, a sixth portion (16.5 g) was added. After about 15 minutes, thereaction mixture was decanted into a separatory funnel and the remainingsolid was rinsed with ethyl acetate (2×1 L). The organics were collectedand washed with 10% sodium bisulfite (1 L). The organic layer was washedwith brine (1 L) and dried over sodium sulfate, filtered andconcentrated. The crude material was purified by flash columnchromatography (1.5 kg silica gel column, gradient elution of 0% to 20%isopropanol in hexanes) to provide the title compound.

Batch 3

(3S,5R,6S)-3-Allyl-5-(3-chlorophenyl)-6-(4-chlorophenyl)-1-((S)-1-(isopropylsulfonyl)-3-methylbutan-2-yl)-3-methylpiperidin-2-one(131.8 g, 239 mmol) was dissolved in acetonitrile (402 mL) and ethylacetate (402 mL) and transferred to a 2 L 3-neck Morton flask. Water(603 mL) was added. The flask was equipped with a thermocouple and amechanical stirrer. Ruthenium(III) chloride hydrate (1.079 g, 4.79 mmol)was added and the reaction vessel was submerged in a cool water bath.Sodium periodate was added in portions (first portion: 59 g), and thetemperature was monitored to keep the reaction mixture below 25° C. Icewas periodically added to the water bath to assist in temperaturecontrol.

After stirring for 45 minutes, a second portion was added (50 g),followed 30 minutes later by a third portion (22 g), after 20 minutes bya forth portion (30 g), and after 20 minutes by a fifth portion (50 g).After stirring for two hours a sixth portion (20 g) was added, followed20 minutes later by a seventh portion (10 g) and 20 minutes after thatby an eighth portion (10 g). After 15 minutes, the reaction mixture wasdecanted into a separatory funnel and the remaining solid was rinsedwith ethyl acetate (2×1 L). The organics were collected and washed with10% sodium bisulfite (1 L). The organic layer was washed with brine (1L) and dried over sodium sulfate, filtered and concentrated. To removeparticulates, the material was dissolved in dichloromethane, filteredand concentrated. The crude material was divided into two portions andeach was purified by flash column chromatography (1.5 kg silica gelcolumn, gradient elution of 0% to 20% isopropanol in hexanes) to providethe title compound.

Batch 4

(3S,5R,6S)-3-Allyl-5-(3-chlorophenyl)-6-(4-chlorophenyl)-1-((S)-1-(isopropylsulfonyl)-3-methylbutan-2-yl)-3-methylpiperidin-2-one(87.3 g, 159 mmol) was dissolved in acetonitrile (302 mL) and ethylacetate (302 mL) and transferred to a 2 L 3-neck Morton flask. Water(453 mL) was added. The flask was equipped with a thermocouple and amechanical stirrer. Ruthenium(III) chloride hydrate (0.786 g, 3.49 mmol)was added and the reaction vessel was submerged in a cool water bath.Sodium periodate was added in portions (first portion: 34.5 g), and thetemperature was monitored to keep the reaction mixture below 25° C. Icewas periodically added to the water bath to assist in temperaturecontrol.

After stirring for 1 hour, a second portion was added (34.4 g), followed30 minutes later by a third portion (34.5 g), and after 30 minutes by aforth portion (34.5 g). The maximum temperature was 27° C. Afterstirring for 3.5 hours a fifth portion (20 g) was added, followed 1 hourlater by a sixth portion (5 g). After 15 minutes, the reaction mixturewas decanted into a reparatory funnel and the remaining solid was rinsedwith ethyl acetate (2×1 L). The organics were collected and washed with10% sodium bisulfite (1 L). The organic layer was washed with brine (0.5L) and dried over sodium sulfate, filtered and concentrated. The crudematerial was purified by flash column chromatography (Biotage SNAP cart,1.5 kg silica gel column, gradient elution of 0% to 50% isopropanol inhexanes) to provide the title compound. Impure fractions were repurifiedby flash column chromatography (1.5 kg silica gel column, gradientelution of 0% to 20% isopropanol in hexanes) to provide the titlecompound.

Batch 5

Impure fractions from Batches 1 through 4 were repurified by multipleiterations of flash column chromatography (amount of silica gel variedfrom 330 g to 1.5 kg, gradient elution of 0% to 20% isopropanol inhexanes) to provide the title compound.

Final Purification

Material from Batches 1 through 5 were combined with a portion of thematerial from another synthesis, 18 g.2-((3R,5R,6S)-5-(3-Chlorophenyl)-6-(4-chlorophenyl)-1-((S)-1-(isopropylsulfonyl)-3-methylbutan-2-yl)-3-methyl-2-oxopiperidin-3-yl)aceticacid (400 g) was dissolved in ethanol and concentrated under a vacuum toprovide a white crystalline solid. A solution of 60% ethanol in water(1900 mL) was added and the mixture was heated to 80° C. while rotatingon a rotary evaporator at atmospheric pressure. After the material haddissolved, the solution was slowly cooled while mechanically stirringthe flask. After 3 hours, the temperature had cooled to 50° C. and thematerial was seeded with crystalline2-((3R,5R,6S)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-1-((S)-1-(isopropylsulfonyl)-3-methylbutan-2-yl)-3-methyl-2-oxopiperidin-3-yl)aceticacid. The solid completely dissolved. After 30 minutes, the solution wasre-seeded (45° C.) and the material began to slowly crystallize. Oncethe mixture had cooled to room temperature, it was placed in the freezerovernight. The crystals were collected by vacuum filtration through aBüchner funnel. The filter cake was washed with ice-cold 60% ethanol inwater and dried under a vacuum on the Büchner funnel to provide a whitesolid. NMR analysis indicated that 7.8 wt % ethanol was present (1 molarequivalent). Water (deionized and filtered (Milli-Q filtration system,EMD Millipore, Billerica, Mass.)) was added to the solid and the mixturewas mechanically stirred at room temperature overnight. Aliquots wereperiodically removed to monitor the ethanol content of the solid. Afterthree days, the material was vacuum filtered through a Büchner funnel,washed with water (deionized and filtered as described above) and driedby pulling a vacuum through the filter cake for 3 hours. The filter cakewas air-dried for two days in the funnel, then, it was transferred to a2 L flask as a white solid and dried under a vacuum overnight. NMRanalysis indicated that 6.2 wt % ethanol was present.

The XRPD pattern was consistent with Compound A ethanolate (FIG. 6).

¹H NMR (500 MHz, DMSO-d₆, δ ppm): 12.43 (br s, 1H), 7.72 (br, 1H), 7.37(br, 2H), 7.23 (t, J=7.8 Hz, 1H), 7.17 (d, J=8.1 Hz, 1H), 7.02 (t,J=1.9, 1.9 Hz, 1H), 6.99 (br, 1H), 6.98 (dt, J=7.7, 1.4, 1.4 Hz, 1H),5.01 (d, J=11.2 Hz, 1H), 3.84 (dd, J=14.0, 10.1 Hz, 1H), 3.59 (ddd,J=13.7, 11.3, 2.9 Hz, 1H), 3.39 (m, 1H), 3.18 (dd, J=13.9, 1.3 Hz, 1H),3.06 (ddd, J=10.6, 8.1, 1.6 Hz, 1H), 2.95 (d, J=13.7 Hz, 1H), 2.50 (d,J=13.8 Hz, 1H), 2.12 (t, J=13.5 Hz, 1H), 2.10 (m, 1H), 2.03 (dd, J=13.3,3.0 Hz, 1H), 1.29 (d, J=6.8 Hz, 3H), 1.29 (d, J=6.8 Hz, 3H), 1.23 (s,3H), 0.55 (d, J=6.6 Hz, 3H), 0.37 (d, J=6.9 Hz, 3H); MS (ESI)=568.2[M+H]⁺.

Synthetic procedures for making2-((3R,5R,6S)-5-(3-chlorophenyl)-6-(4-chlorophenyl)-1-((S)-1-(isopropylsulfonyl)-3-methylbutan-2-yl)-3-methyl-2-oxopiperidin-3-yl)aceticacid (Compound A)

Preparation of Propane-2-Sulfinic Acid

Tetrahydrofuran (20 L) was added to a reaction vessel and thetemperature of the vessel was cooled to −50° C. Sulfur dioxide (3.5 kg,54.6 mol) was condensed in the reaction vessel at −50° C. Isopropylmagnesium chloride (2M in tetrahydrofuran, 21 L, 42 mol) was added tothe solution. The reaction mixture was agitated for 30 min at −10° C.and aqueous 2.5 N hydrochloric acid (18.5 l, 46.2 mol) was added. Thereaction mixture was warmed to 20° C. and t-butylmethyl ether (10 L) wasadded. The phases were separated and the aqueous phase was extractedtwice with t-butylmethyl ether (10 L). The combined organic extractswere washed with aqueous sodium chloride (12 wt %, 20 mL) andconcentrated under reduced pressure to afford the desired sulfinic acidin 82% yield (3.7 Kg).

Preparation of(3S,5R,6S)-3-allyl-5-(3-chlorophenyl)-6-(4-chlorophenyl)-1-((S)-1-(isopropylsulfonyl)-3-methylbutan-2-yl)-3-methylpiperidin-2-one

To a solution of propane-2-sulfinic acid (912 g, 8.4 mol) in toluene(7.5 L) was added tetrahydrofuran (3.6 L). Sodium t-butoxide (2M intetrahydrofuran, 3.6 L, 7.2 mol) was added while maintaining thetemperature of the mixture below 20° C. The pH of the mixture wasmeasured to be approximately 6. The mixture was distilled underatmospheric pressure to produce a distillate mass of 6.6 Kg.(3S,5S,6R,8S)-8-Allyl-6-(3-chlorophenyl)-5-(4-chlorophenyl)-3-isopropyl-8-methyl-2,3,5,6,7,8-hexahydrooxazolo[3,2-a]pyridin-4-iumnaphthalene-1-sulfonate, hemi-toluene solvate (also called the“oxoiminium salt, hemi-toluene solvate” herein) (3.62 Kg, 5.2 mol) andtoluene (7.8 L) were added, maintaining the temperature of the mixturebelow 30° C. The mixture was distilled under atmospheric pressure toproduce a distillate mass of 7.2 Kg while simultaneously addingdimethylacetamide (10.9 L). The mixture was agitated at approximately120° C. for 14 h and cooled to 25° C. t-Butylmethyl ether (9.1 L) andwater (14.5 L) were added to the mixture and the biphasic mixture wasagitated until no solids were visible. The phases were separated. Theorganic phase was washed with water (7.3 L) and aqueous saturated sodiumbicarbonate (7.1 L). The organic phase was filtered and distilled underreduced pressure to produce a distillate mass of 15 Kg whilesimultaneously adding acetonitrile (21.3 L). Water (2 L) was added andthe solution was seeded with(3S,5R,6S)-3-allyl-5-(3-chlorophenyl)-6-(4-chlorophenyl)-1-((S)-1-(isopropylsulfonyl)-3-methylbutan-2-yl)-3-methylpiperidin-2-one(160 g, 0.29 mol) at 25° C. (The seed material was prepared via the sameprocess in a previously conducted smaller scale experiment). The mixturewas agitated at 25° C. for 25 min and cooled to 20° C. overapproximately 45 min. A mixture of acetonitrile (3.0 L) and water (7.0L) was added to the reaction mixture over 1.5 h. The resultant mixturewas agitated for 1 h and filtered. The product was washed with a mixtureof acetonitrile (3.6 L) and water (2.4 L). The product was dried undernitrogen to afford(3S,5R,6S)-3-allyl-5-(3-chlorophenyl)-6-(4-chlorophenyl)-1-((S)-1-(isopropylsulfonyl)-3-methylbutan-2-yl)-3-methylpiperidin-2-one(2.9 Kg) in 86% yield.

Preparation of Compound A Ethanolate

To a solution of(3S,5R,6S)-3-allyl-5-(3-chlorophenyl)-6-(4-chlorophenyl)-1-((S)-1-(isopropylsulfonyl)-3-methylbutan-2-yl)-3-methylpiperidin-2-one(2.4 Kg, 4.4 mol) in ethyl acetate (8.4 L), acetonitrile (8.6 L), andwater (6.5 L) was added ruthenium chloride hydrate (20.5 g, 0.09 mol).Sodium periodate (5.0 kg, 23.2 mol) was added in four 4 equal portionsover the course of 1.5 h, maintaining the temperature of the mixturebetween 20° C. and 28° C. The mixture was agitated for 2.5 h andfiltered through a layer of diatomaceous earth (3.33 Kg). The resultingdiatomaceous earth cake was washed with isopropyl acetate (10.4 L) andwater (3 L). The filtrate was phase separated. The organic phase waswashed twice with an aqueous sodium chloride solution (25 wt %, 5.5 L),washed twice with an aqueous sodium chloride and sodium bisulfitesolution (25 wt % sodium chloride and 20 wt % sodium bisulfite, 7.8 L),and once with an aqueous sodium chloride solution (25 wt %, 6.5 L). Theorganic phase was distilled under reduced pressure while simultaneouslyadding isopropyl acetate (12.4 L). The batch was filtered. Charcoal (680g) was added and the mixture was agitated for 13 h. The mixture wasfiltered through a layer of diatomaceous earth (1.5 Kg) and thediatomaceous earth cake was washed with isopropyl acetate (8 L). Thesolution was distilled under reduced pressure to produce a distillatemass of 24.5 Kg while simultaneously adding ethanol (16 L). Heptane (8.5L) was added and the solution was seeded with Compound A Ethanolate (Theseed material was prepared via the same process in a previouslyconducted smaller scale experiment) (95 g). The mixture was agitated at20° C. for 40 min and distilled under reduced pressure to produce adistillate mass of 10.9 Kg while simultaneously adding heptane (8.8 L).The mixture was agitated for 12 h and filtered. The product was washedwith a mixture ethanol (0.4 L) and heptane (1.6 L). The product wasdried under nitrogen to afford Compound A Ethanolate (1.99 Kg) in 70%yield.

Preparation of Compound A Crystalline Anhydrous

Compound A Ethanolate (1.0 Kg, 1.62 mol) was dissolved in methanol (8.5L) and the resultant solution was filtered. The solution was warmed to35° C. and water (2.5 L) was added. The solution was seeded withCompound A Crystalline Anhydrous (50 g, 0.074 mol) and cooled to 20° C.over the course of 4 h (The seed material was prepared via the sameprocess in a previously conducted smaller scale experiment). Water (2 L)was added over the course of 30 min. The mixture was agitated for 30 minand filtered. The product was dried under nitrogen to afford Compound ACrystalline Anhydrous (0.86 Kg) in 93% yield.

¹H NMR (400 MHz, DMSO-d₆) δ 12.37 (s, 1H), 7.36 (bs, 4H), 7.23 (t, 1H,J=7.9 Hz), 7.16 (ddd, 1H, J=7.9, 1.9, 1.0 Hz), 7.02 (t, 1H, J=1.9 Hz),6.98 (bd, 1H, J=7.9 Hz), 5.02 (d, 1H, J=7.9 Hz), 3.84 (dd, 1H, J=13.4,10.2 Hz), 3.58 (ddd, 1H, J=13.5, 11.3, 3.0 Hz), 3.39 (spt, 1H, J=6.8Hz), 3.17 (bd, 1H, J=13.4 Hz), 3.07 (bt, 1H, J=8.6 Hz), 2.95 (d, 1H,J=13.9 Hz), 2.51 (d, 1H, J=13.9 Hz), 2.13 (bt, 1H, J=13.5 Hz), 2.11(spt, 1H, J=6.8 Hz), 2.04 (dd, 1H, J=13.5, 3.0 Hz), 1.30 (2×d, 6H, J=6.8Hz), 1.24 (s, 3H), 0.56 (d, 3H, J=6.8 Hz), 0.38 (d, 3H, J=6.8 Hz); ExactMass [C₂₈H₃₆Cl₂NO₅S]⁺: calculated=568.1691, measured M/Z [M+1]=568.1686.

It is noted that when seed crystals are used in the procedures set forthin this application, the seed crystals can be obtained by following theprocedures set forth herein, typically on a smaller scale, to obtainseed crystals for the larger scale syntheses.

Preparation of Calcium Propane-2-Sulfinate Dihydrate

Tetrahydrofuran (20 L) was added to a reaction vessel and thetemperature of the vessel was cooled to −50° C. Sulfur dioxide (3.5 kg,54.6 mol) was condensed in the reaction vessel at −50° C. Isopropylmagnesium chloride (2M in tetrahydrofuran, 21 L, 42 mol) was added tothe solution. The reaction mixture was agitated for 30 min at −10° C.and aqueous 2.5 N hydrochloric acid (18.5 l, 46.2 mol) was added. Thereaction mixture was warmed to 20° C. and t-butylmethyl ether (10 L) wasadded. The phases were separated and the aqueous phase was extractedtwice with t-butylmethyl ether (10 L). The combined organic extractswere washed with aqueous sodium chloride (12 wt %, 20 mL) andconcentrated under reduced pressure to afford the desiredpropane-2-sulfinic acid in 82% yield (3.7 Kg). The propane-2-sulfinicacid was dissolved in ethanol (37 L) and a solution of calcium acetatemonohydrate (3.0 Kg, 17.1 mol) in water (7.2 L) was added. The resultantmixture was agitated for 1 h and filtered. The product was washed with amixture of ethanol (10.8 L) and water (1.1 L). The product was driedunder nitrogen to afford calcium propane-2-sulfinate dihydrate in 86%yield (4.26 Kg). ¹H NMR (400 MHz, DMSO-d6) δ 3.37 (s, 4H), 1.88 (spt,2H, J=7.0 Hz), 0.92 (d, 12H, J=7.0 Hz). Preparation of(3S,5R,6S)-3-allyl-5-(3-chlorophenyl)-6-(4-chlorophenyl)-1-((S)-1-(isopropylsulfonyl)-3-methylbutan-2-yl)-3-methylpiperidin-2-one

Calcium propane-2-sulfinate dihydrate (2943616) (2.7 Kg, 9.36 mol) andtoluene (22 L) were added to a 60 L vessel. The reaction mixture waswarmed to 110° C. and distilled under reduced pressure to produce adistillate mass of 50 Kg while simultaneously adding toluene (43 L). Thereaction mixture was cooled to 40° C. and(3S,5S,6R,8S)-8-Allyl-6-(3-chlorophenyl)-5-(4-chlorophenyl)-3-isopropyl-8-methyl-2,3,5,6,7,8-hexahydrooxazolo[3,2-a]pyridin-4-iumnaphthalene-1-sulfonate, hemi toluene solvate (3.6 Kg, 5.2 mol) andtoluene (9.0 L) were added. The reaction mixture was warmed to 110° C.and distilled under atmospheric pressure to produce a distillate mass of15.8 Kg while simultaneously adding dimethylacetamide (10.9 L). Themixture was agitated at approximately 120° C. for 14 h and cooled to 40°C. t-Butylmethyl ether (9.1 L) and water (14.5 L) were added to themixture and the biphasic mixture was agitated until no solids werevisible. The phases were separated. The organic phase was washed twicewith water (2×7.3 L), once with aqueous saturated sodium bicarbonate(7.1 L), and once with an aqueous sodium chloride (12 wt %, 7.1 L). Theorganic phase was cooled to 20° C., filtered, and distilled underreduced pressure to produce a distillate mass of 15 Kg whilesimultaneously adding acetonitrile (21.3 L). Water (2 L) was added. Thesolution was seeded with(3S,5R,6S)-3-allyl-5-(3-chlorophenyl)-6-(4-chlorophenyl)-1-((S)-1-(isopropylsulfonyl)-3-methylbutan-2-yl)-3-methylpiperidin-2-one(160 g, 0.29 mol) at 25° C. The mixture was agitated at 25° C. for 25min and cooled to 20° C. over approximately 45 min (The seed materialwas prepared via the same process in a previously conducted smallerscale experiment). A mixture of acetonitrile (3.0 L) and water (7.0 L)was added to the reaction mixture over 1.5 h. The resultant mixture wasagitated for 1 h and filtered. The product was washed with a mixture ofacetonitrile (3.6 L) and water (2.4 L). The product was dried undernitrogen to afford(3S,5R,6S)-3-allyl-5-(3-chlorophenyl)-6-(4-chlorophenyl)-1-((S)-1-(isopropylsulfonyl)-3-methylbutan-2-yl)-3-methylpiperidin-2-one(2.8 Kg) in 83% yield.

Preparation of Compound A Ethanolate

A solution of(3S,5R,6S)-3-allyl-5-(3-chlorophenyl)-6-(4-chlorophenyl)-1-((S)-1-(isopropylsulfonyl)-3-methylbutan-2-yl)-3-methylpiperidin-2-one(1.6 Kg, 2.9 mol) in a mixture of water (2.4 L) and acetonitrile (21.6L) was allowed to flow through the continuous stirred-tank reactor ozonevessel (1 L vessel) at a flow rate of 60 mL/min at 20° C.(alternatively, the ozonolysis was performed in a reaction vessel usingan ozone sparger). The reaction mixture was added to a solution ofsodium chlorite (80 wt %, 1.0 Kg, 11.6 mol) in water (5.6 L) over thecourse of 6 h (alternatively, the aqueous solution of sodium chloritewas added to the reaction mixture). The reaction mixture was agitatedfor 16 h and a solution of sodium bisulfite (1.2 Kg, 11.6 mol) in water(5.6 L) was added over the course of 2 h. The mixture was agitated for 1h and the phases were separated. To the organic phases were addedisopropyl acetate (8 L) and water (8 L). The mixture was agitated for 30min and the phases were separated. The organic phase was washed oncewith aqueous sodium chloride (6 wt %, 8 L), three times with aqueous 1Msodium phosphate (pH 6, 8 L), and once with aqueous sodium chloride (6wt %, 8 L). The organic phase was filtered. The mixture was distilledunder reduced pressure to produce a distillate mass of 35 Kg whilesimultaneously adding isopropyl acetate (32 L). The mixture wasdistilled under reduced pressure to produce a distillate mass of 36 Kgwhile simultaneously adding ethanol (32 L). Heptane was added (9.6 L)and the mixture was distilled under reduced pressure to produce adistillate mass of 5 Kg. The mixture was seeded with Compound AEthanolate (80 g, 0.13 mol) (The seed material was prepared via the sameprocess in a previously conducted smaller scale experiment). Heptane(6.4 L) was added over the course of 1 h, the mixture was agitated for12 h, cooled to 15° C., and filtered. The product was washed with amixture of ethanol (90 mL) and heptane (4.8 L). The product was driedunder nitrogen to afford Compound A Ethanolate (1.33 Kg) in 81% yield.

Preparation of Compound A Crystalline Anhydrous

Compound A Ethanolate (1.0 Kg, 1.62 mol) was dissolved in methanol (8.5L) and the resultant solution was filtered. The solution was warmed to35° C. and water (2.5 L) was added. The solution was seeded withCompound A Crystalline Anhydrous (50 g, 0.074 mol) and cooled to 20° C.over the course of 4 h (The seed material was prepared via the sameprocess in a previously conducted smaller scale experiment). Water (2 L)was added over the course of 30 min. The mixture was agitated for 30 minand filtered. The product was dried under nitrogen to afford Compound ACrystalline Anhydrous (0.86 Kg) in 93% yield.

¹H NMR (400 MHz, DMSO-d₆) δ 12.37 (s, 1H), 7.36 (bs, 4H), 7.23 (t, 1H,J=7.9 Hz), 7.16 (ddd, 1H, J=7.9, 1.9, 1.0 Hz), 7.02 (t, 1H, J=1.9 Hz),6.98 (bd, 1H, J=7.9 Hz), 5.02 (d, 1H, J=7.9 Hz), 3.84 (dd, 1H, J=13.4,10.2 Hz), 3.58 (ddd, 1H, J=13.5, 11.3, 3.0 Hz), 3.39 (spt, 1H, J=6.8Hz), 3.17 (bd, 1H, J=13.4 Hz), 3.07 (bt, 1H, J=8.6 Hz), 2.95 (d, 1H,J=13.9 Hz), 2.51 (d, 1H, J=13.9 Hz), 2.13 (bt, 1H, J=13.5 Hz), 2.11(spt, 1H, J=6.8 Hz), 2.04 (dd, 1H, J=13.5, 3.0 Hz), 1.30 (2×d, 6H, J=6.8Hz), 1.24 (s, 3H), 0.56 (d, 3H, J=6.8 Hz), 0.38 (d, 3H, J=6.8 Hz); ExactMass [C₂₈H₃₆Cl₂NO₅S]⁺: calculated=568.1691, measured M/Z [M+l]=568.1686.An XRPD pattern representative of compound A crystalline anhydrous isshown in FIG. 1.

An alternative route to a make compound A crystalline anhydrous is tomake a DABCO salt instead of the ethanolate as shown in Scheme 3.

Preparation of Compound A DABCO Salt

Ozone was delivered to an agitated solution of(3S,5R,6S)-3-allyl-5-(3-chlorophenyl)-6-(4-chlorophenyl)-1-((S)-1-(isopropylsulfonyl)-3-methylbutan-2-yl)-3-methylpiperidin-2-one(4.0 Kg, 7.27 mol) in a mixture of water (6 L) and acetonitrile (54 L)using a subsurface C22 Hastelloy sparger at 20° C. over the course often hours. An aqueous solution of sodium chlorite (80 wt %, 2.5 Kg, 29mol) in water (14 L) was added over the course of 1 h, maintaining thetemperature of the mixture below 40° C. The reaction mixture wasagitated for 12 h and a solution of sodium bisulfite (3.0 Kg, 29 mol) inwater (14 L) was added over the course of 2 h, maintaining thetemperature of the reaction mixture below 40° C. The mixture wasagitated for 1 h and the phases were separated. To the organic phaseswere added isopropyl acetate (IPAC) (20 L) and 1M aqueous sodiumphosphate pH 6 (8 L). The mixture was agitated for 30 min and the phaseswere separated. The organic phase was washed with 1M aqueous sodiumphosphate pH 6 (20 L) and with 1M aqueous sodium chloride (20 L). Themixture was distilled under reduced pressure to produce a distillatemass of 75 Kg while simultaneously adding isopropyl acetate (80 L). Thewater content of the solution by Karl Fisher was less than one percent.The organic phase was filtered. The solution was further distilled to avolume of approximately 16 L. The solution was heated to 55° C. and1,4-diazabicyclo[2.2.2]octane (DABCO, 424 g, 3.65 mol) was added.(3S,5R,6S)-3-allyl-5-(3-chlorophenyl)-6-(4-chlorophenyl)-1-((S)-1-(isopropylsulfonyl)-3-methylbutan-2-yl)-3-methylpiperidin-2-one1,4-diazabicyclo[2.2.2]octane (DABCO) salt seeds (136 g, 0.18 mol) wereadded as a slurry in isopropyl acetate and heptane (1/1, 800 mL). Themixture was agitated at 55° C. for 20 minutes and cooled to 20° C. overthe course of 2 h. Heptane was added (16.8 L) over the course of 1 h andthe mixture was agitated at 20° C. for 12 h. The product was filteredand the filter cake was washed once with a mixture of isopropyl acetateand heptane (⅔, 21 L) and once with a mixture of isopropyl acetate andheptane (¼, 21 L). The product was dried under nitrogen to affordCompound A DABCO Salt (4.64 Kg) in 87% yield (100% liquid chromatographyarea percent (LCAP), 78.9 wt % Compound A). The compound A DABCO salt isa solvate of isopropyl acetate (IPAC) in accordance with Scheme 3. TheCompound A DABCO Salt is the better performing purification controlpoint to enhance the purity of the drug substance (Compound A).Typically, the purity of crude reaction mixtures of 97 to 99 liquidchromatography area percent purity can be improved to 100 liquidchromatography area percent purity (no impurity at greater level than0.05 liquid chromatography area percent) using the crystallization ofthe DABCO salt. For comparison, enhancement of purity of the drugsubstance (Compound A) using Compound A Ethanolate as a control pointallows for crude reaction mixtures of 97 to 99 liquid chromatographyarea percent purity to be improved to 99.5 to 99.6 liquid chromatographyarea percent purity (and multiple impurities are present in the filteredmaterial at greater levels than 0.05 liquid chromatography areapercent).

¹H NMR (400 MHz, CDCl₃): δ ppm 0.49 (d, J=6.8 Hz, 6H), 0.64 (d, J=6.4Hz, 6H), 1.23 (d, J=6.0 Hz, 12H), 1.41 (s, 6H), 1.43 (d, J=7.6 Hz, 12H),2.02 (s, 6H), 2.05-2.00 (m, 2H), 2.30-2.15 (m, 4H), 2.71 (d, J=13.2,2H), 2.84 (dd, J=2.0, 13.6, 2H), 2.90 (d, J=13.6 Hz, 2H), 2.96 (s, 12H),3.11 (pent, J=6.8 Hz, 2H), 3.67-3.22 (m, 2H), 3.55-3.48 (m, 2H), 4.07(dd, J=10.4, 13.2 Hz, 2H), 4.99 (sept, J=6.4 Hz, 2H), 5.13 (d, J=11.2Hz, 2H), 7.10-6.98 (m, 8H), 7.35-7.10 (m, 8H), 13.2 (br, 2H). ¹³C NMR(101 MHz, CDCl₃) δ ppm 15.3, 15.7, 20.3, 21.0, 21.4, 21.8, 25.6, 32.6,39.6, 41.5, 44.5, 44.6, 44.8, 47.0, 54.8, 58.4, 67.6, 69.2, 76.7, 77.0,77.4, 125.7, 126.9, 128.2, 128.5, 129.8, 133.9, 134.0, 137.5, 143.8,170.7, 174.6, 176.3. m.p. 103° C.

Preparation of Compound A Crystalline Anhydrous

To(3S,5R,6S)-3-allyl-5-(3-chlorophenyl)-6-(4-chlorophenyl)-1-((S)-1-(isopropylsulfonyl)-3-methylbutan-2-yl)-3-methylpiperidin-2-one1,4-diazabicyclo[2.2.2]octane (DABCO) salt (8.28 Kg, 5.79 mol) wereadded isopropyl acetate (41.4 L) and water (41.4 L). To the mixture wasadded 4M aqueous hydrochloric acid (3 L, 12.1 mol) and the biphasicmixture was agitated for 30 minutes. The phases were separated and theorganic phases was washed twice with 1M aqueous sodium phosphate pH 6(25 L) and once with aqueous sodium chloride (7 wt %, 33 L). The mixturewas distilled under reduced pressure to produce a distillate mass of 56Kg while simultaneously adding isopropyl acetate (42 L). The isopropylacetate content and the water content by Karl Fisher were both measuredto be less than one percent in the solution. The organic phase wasfiltered. The organic phase was distilled under reduced pressure togenerate a distillate mass of 20 kg while simultaneously adding aceticacid (45 L). The solution was heated to 60° C. and deionized water (29L) was added over the course of 30 minutes.(3S,5R,6S)-3-allyl-5-(3-chlorophenyl)-6-(4-chlorophenyl)-1-((S)-1-(isopropylsulfonyl)-3-methylbutan-2-yl)-3-methylpiperidin-2-oneseeds (320 g, 0.56 mol) were added as a slurry in acetic acid anddeionized water (3/2, 1 L). The mixture was agitated at 60° C. for 3 h,and cooled to 20° C. over the course of 6 h. The mixture was agitated at20° C. for 12 h. Deionized water (7 mL) was added over the course of 1 hand the mixture was agitated for one additional hour. The product wasfiltered and the filter cake was washed once with a mixture of aceticacid and deionized water (1/1, 13 L) and three times with deionizedwater (3×65 L). The product was dried under nitrogen to afford(3S,5R,6S)-3-allyl-5-(3-chlorophenyl)-6-(4-chlorophenyl)-1-((S)-1-(isopropylsulfonyl)-3-methylbutan-2-yl)-3-methylpiperidin-2-one (6.3 Kg) in 92%yield (100% LCAP, 100.3 wt %, 320 ppm acetic acid, <100 ppm water).

A synthesis of Compound A is shown in Scheme A. An importantintermediate in the synthesis is the compound(3S,5S,6R,8S)-8-Allyl-6-(3-chlorophenyl)-5-(4-chlorophenyl)-3-isopropyl-8-methyl-2,3,5,6,7,8-hexahydrooxazolo[3,2-a]pyridin-4-iumnaphthalene-1-sulfonate (also called the “oxoiminium salt” or“oxazolinium salt” herein). Due to difficulties crystallizing the TfO⁻or TsO⁻ salts of(3S,5S,6R,8S)-8-Allyl-6-(3-chlorophenyl)-5-(4-chlorophenyl)-3-isopropyl-8-methyl-2,3,5,6,7,8-hexahydrooxazolo[3,2-a]pyridin-4-iumnaphthalene-1-sulfonate, they were not isolated. Crystallization isuseful because it can be used to remove impurities generated in theprocess or found in the starting materials. Hence, a hydrolysis to acrystalline lactam followed by a re-formation of the oxoiminium salt canbe used.

The present invention describes a process to make an oxoiminiumnaphthalenesulfonate salt, and particularly an oxoiminiumnaphthalenesulfonate salt, hemi toluene solvate, that is crystalline.Using the oxoiminium naphthalenesulfonate salt, hemi-toluene solvateprovides for an improved method of making Compound A (See, Scheme Bbelow).

The oxoiminium salt, hemi-toluene hydrate was made by heating(3S,5R,6S)-3-allyl-5-(3-chlorophenyl)-6-(4-chlorophenyl)-1-((S)-1-hydroxy-3-methylbutan-2-yl)-3-methylpiperidin-2-oneand 1-naphthalene sulfonic acid in toluene under dehydrative conditions.The crystalline material is characterized as a hemi-toluene solvate byNMR, DSC, and XRPD. This crystalline form is a shelf-stable substance,which is, therefore, well suited as a reagent to make Compound A. Oneway of making the oxoiminium salt is by ion exchange using1-naphthalene-sulfonate, followed by crystallization from toluene. Itwas found that the advantages of using 1-naphthalene sulfonate overother counterions included rapid crystallization kinetics, predictablecrystal habit and size, low room-temperature solubility in toluene (<10mg/ml), high melting point (207-209° C.), and most importantly, highimpurity purging capability. All process impurities includingstereoisomers were routinely purged to less than 0.5 liquidchromatography area percent (LCAP) with a single crystallization. (SeeScheme C below)

Formation of the oxoiminium salt as shown in Scheme D below could beaccomplished by double dehydrative cyclization using Tf₂O undercryogenic conditions (conditions a) or using Ts₂O at elevatedtemperatures (conditions b).

The advantages of Conditions a is that the reaction could be performedin a single step. However, these conditions can have side reactions(such as undesirable elimination leading to stilbene-type byproducts)and undesirable cryogenic processing. The latter (conditions b) is astep-wise process, with well characterized formation of intermediates onroute to the oxoiminium naphthalenesulfonate salt. Since Ts₂O is amilder reagent, undesirable double cyclizations are significantlyreduced and higher yields (>75% vs <60% yield) can be obtained. Inaddition, the process is more desirable for scale-up under heatingconditions.

Step-wise conversion of valinol adduct (labeled “amide” in Scheme E) tooxoiminium naphthalenesulfonate salt under Ts₂O conditions is shown inScheme E.

Below is the a description of the process that enabled multiple kilogramdelivery of the oxoiminium salt. The first step of the process isreacting(3S,5R,6R)-3-allyl-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyltetrahydro-2H-pyran-2-onewith L-valinol at an elevated temperature. The low optical purity (80%ee) and general purity (85%) of the starting lactones is acceptable. Thevalinol adduct is formed as a diastereomeric mixture, which istelescoped into subsequent synthetic steps.

In the presence of 2,6-lutidine, the reaction of the valinol adduct(amide in Scheme E) with tosic anhydride is essentially instantaneous at15 to 25° C., providing hydroxy oxazoline as a stable intermediate. Inthe presence of additional tosic anhydride and 2,6-lutidine, a secondobservable reaction intermediate, tosyl oxazoline, forms. Finally, afterprolonged heating of the reaction mixture at its reflux temperature (55°C. for 1 day), the reaction proceeds to completion to provide oxoiminiumtosylate.

The reaction mixture is quenched with sulfuric acid and washed multipletimes with a sodium 1-naphthylsulfonate solution to facilitate counterion exchange. After a distillation step in which the reaction solvent isswitched from dichloromethane to toluene, oxoiminium salt crystallizesas a rod-like hemi-toluene solvate.

In summary, crystalline oxoiminium salt is an isolatable, stableintermediate that is good for purging various impurities such asdiastereomers and stilbene using crystallization. As a material to makecompound A, the oxoiminium salt, hemi-toluene solvate has desirablefeatures, including isolability in high chemical and stereoisomericpurity, bulk properties suitable for standard manufacturing techniques,and stability to storage.

Preparation of Oxoiminium Salt, Hemi-Toluene Solvate

In accordance with Scheme F, L-Valinol (2.6 Kg, 25.2 mol) was melted at50° C. and(3S,5R,6R)-3-allyl-5-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyltetrahydro-2H-pyran-2-one(3.6 Kg, 84.0 wt %, 80.8% ee, 7.9 mol) was added. The mixture was heatedto 110° C. and agitated at that temperature for 5 h. The mixture wascooled to 20° C. and dichloromethane (17.9 L) was added. Aqueous 1Nhydrochloric acid (18.5 L) was added and the biphasic mixture wasagitated for 10 min. The phases were separated and the organic phase waswashed with an aqueous sodium chloride solution (20 wt %, 7 L). Theorganic phase was distilled under atmospheric pressure to produce adistillate mass of 13.7 Kg while simultaneously adding dichloromethane(3.3 L). The organic phase was added over the course of 10 min to asolution of p-toluene sulfonic anhydride (5.9 Kg, 18 mol) indichloromethane (23.0 L). 2,6-Lutidine (3.56 Kg, 33.2 mol) was addedover the course of 1 h, maintaining the temperature of the mixture below25° C. The mixture was agitated at 20° C. for 40 min. The mixture wasdistilled under atmospheric pressure and at 40° C. to produce adistillate mass of 13.0 Kg. The mixture was added to aqueous 2N sulfuricacid (19.5 Kg) over the course of 15 min, maintaining the temperaturebelow 20° C. The mixture was agitated for 15 min and the phases wereseparated. The organic phase was washed twice with an aqueous sodium1-naphthylsulfonate solution (10 wt %, 19.4 Kg), and once with anaqueous sodium bicarbonate solution (5 wt %, 19.5 Kg).1-naphthylsulfonic acid dihydrate (64 g, 0.26 mol) was added.

The organic phase was distilled under reduced pressure and maintaining atemperature of 50° C. to produce a distillate mass of 39.9 Kg whilesimultaneously adding toluene (27.0 L). The mixture was seeded withoxoiminium salt, hemi-toluene solvate (40 g, 0.06 mol) and agitated for20 min (The seed material was prepared via the same process in apreviously conducted smaller scale experiment). The mixture was cooledto 20° C. and agitated for 20 h. The mixture was filtered. The productcake was washed with toluene (7.9 L) and dried under nitrogen to affordoxoiminium salt, hemi-toluene solvate (3.7 Kg, 63.6 wt %, 99.7% ee, 99/1DR) in 76% yield.

¹H NMR (400 MHz, DMSO-d₆) δ 8.03-8.00 (m, 1H), 7.93-7.90 (m, 3H),7.56-7.42 (m, 6.5H), 7.33 (s, 1H), 7.27-7.13 (m, 6H), 5.85 (m, 1H), 5.35(m, 3H), 5.02 (m, 1H), 4.93 (t, 1H, J=9.98 Hz), 4.3 (m, 1H), 4.09 (m,1H), 2.79 (m, 2H), 2.39 (t, 1H, J=13.3 Hz), 2.3 (s, 1.5H), 2.01 (dd, 1H,J=13.69, 3.13 Hz), 1.34 (s, 3H), 0.61 (d, 3H, J=6.46 Hz), 0.53 (d, 3H,J=6.85 Hz), 0.41 (m, 1H)

Anhydrous Oxoiminium Salt

The oxoiminium salt, hemi-toluene solvate (1 g) was dissolved inchloroform (10 mL) and the solution was concentrated under reducedpressure. To the residue obtained was added chloroform (10 mL) and thesolution was concentrated under reduced pressure again. Finally, to theresidue obtained was added chloroform (10 mL) and the solution wasconcentrated under reduced pressure.

¹H NMR (400 MHz, CDCl₃) δ 9.13 (d, 1H, J=8.61 Hz), 8.35 (d, 1H, J=7.24Hz), 7.86 (t, 2H, J=9.0 Hz), 7.57 (m, 1H), 7.48 (m, 2H), 7.28 (m, 5H),7.09 (m, 3H), 6.11 (d, 1H, J=11.15 Hz), 5.81 (m, 1H), 5.54 (m, 1H), 5.32(m, 2H), 4.79 (m, 1H), 4.64 (dd, 1H, J=9.00, 4.89 Hz), 3.56 (m, 1H),2.89 (t, 1H, J=13.69 Hz), 2.65 (m, 2H), 1.97 (dd, 1H, J=14.08, 3.33 Hz),1.54 (s, 3H), 0.66 (s, 3H), 0.36 (m, 1H), 0.59 (s, 3H)

1. Crystalline

2.-38. (canceled)